Tag: hydroponic

A common pathway in most organisms for detoxifying xenobiotics is through glutathione conjugation catalyzed by glutathione Stransferases . It has long been recognized that GSTs play an important role in normal cellular metabolism in plants . In addition, GST mRNA responds quickly to exposure of xenobiotics , protecting the cell against oxidative stress or chemical toxicity . Previous research on xenobiotics such as herbicides shows that differences in GSH availability and in the portfolio of GST isoenzymes are associated with xenobiotic resistance . Conjugation with various biomolecules was observed in plant metabolism of different PPCPs, including diclofenac , ibuprofen , triclosan , benzotriazole and acetaminophen . However, GST-mediated conjugation may play a predominant role, but has been relatively understudied so far . In particular, the entire process of GST mediated detoxification, from GSH conjugation in the cytoplasm to vacuolar accumulation and processing of conjugates has yet to be elucidated in vivo. Acetaminophen and structural analogs are phenolic compounds that are among ubiquitous environmental contaminants . Acetaminophen is one of the most used pain and fever relief medicines, and many billions of doses of acetaminophen are consumed each year . Acetaminophen was detected in the aquatic environment at 0.01–0.3 mg L−1 in the South Wales region of the UK .Cucumber is a crop plant, widely consumed, with high economic and ecological relevance,hydroponic grow system and recommended for use in phytotoxicity studies by U.S. Environmental Protection Agency . In this study, we used acetaminophen to elucidate the mechanisms of GST-mediated detoxification in cucumber plants.

Findings of this study may provide evidence for PPCP detoxification via GSH conjugation, and will likely prompt further exploratory investigation on the role and value of this pathway for the numerous emerging contaminants.Cucumber seeds were obtained from Fisher and transferred to a 72-hole plate filled with a mixture of vermiculite and perlite . Plants were cultivated under controlled conditions for 1 week and irrigated with half strength modified Hoagland nutrient solution, which is adequate for the seedlings’ energy demands for cucumber growth and the low ionic strength nutrient solution permits a more precise computation of acetaminophen speciation than do more complete nutrient medium. Details on the solution composition are given in Table S2. Seedlings in the third-leaf stage were removed from the pots, rinsed with tap water, and placed in 500 mL glass jars containing aerated, half strength modified Hoagland nutrient solution. Each treatment contained three replicates, and each replicate was prepared from a single plant by separating into roots and shoots.After 7 days of adaption, plants were exposed to acetaminophen by spiking 25 μL stock solution to yield an initial concentration of 5.0 mg L−1 .Simultaneously, treatment blanks and plant blanks were included in duplicate to determine abiotic losses of acetaminophen under the experimental conditions. All experimental materials were auto-claved, 121 °C for 30 min, before use. A parallel group trial enrolled with acetaminophen at 60 μg L−1 was included to simulate more realistic levels and to validate the high level treatment. The cultivation lasted for 20 days, with the nutrient solution renewed every other day . Additionally, in order to determine if plants can detoxify acetaminophen via GSH conjugation in realistic field environment, cucumber seedlings were cultivated for 20 days in soil amended with 5% bio-solids containing acetaminophen at an initial concentration of 10 mg kg−1 .

A root exudate control was included to evaluate the effect of plant exudates on microbial degradation and GSH conjugation of acetaminophen in the hydroponic solution. Cucumber seedlings were cultivated in 500 mL glass jars containing aerated, half strength modified Hoagland nutrient solution, and the plants were removed after 7 days. The solution was brought back to 500 mL with half strength modified Hoagland nutrient solution, and then 25 μL of acetaminophen stock solution was added to yield an initial concentration of 5.0 mg L−1 . The nutrient solution samples were exposed to the same conditions, and were analyzed for acetaminophen and its conjugates. To investigate whether GSH-acetaminophen can be transported from the root to the shoot, two-week-old cucumber plants were exposed to 1 mg L−1 GSH-acetaminophen for 2 days. Leaves were collected and extracts from the aerial parts were analyzed as described below. To test whether cytochrome P450 was involved in GSH-conjugates formation, two P450 inhibitors, 1-aminobenzotriazole and piperonyl butoxide, were used to pretreat the plant on the basis of previous studies demonstrated specific inhibition effects . Cucumber seedlings were pretreated with 1- aminobenzotriazole or piperonyl butoxide for 12 h in 500 mL glass jars with aerated, half strength modified Hoagland nutrient solution, followed by spiking 25 μL of acetaminophen stock solution to yield an initial concentration of 5.0 mg L−1 . After 96 h incubation, tissue samples were collected from both the treated and untreated plants.The specificity of these P450 inhibitors was evaluated by monitoring cytochrome P450 activity, GSH content and GST activity in cucumber tissues after the treatment. For better clarity, all the experiments performed was schematically represented Table S3. Simultaneously, commercially available common bean , tomato , alfalfa and wheat seeds were germinated and transplanted under the same conditions as above. After 7 days of adaption, plants were exposed to acetaminophen at 5.0 mg L−1 . After 96 h, plant roots were collected and analyzed for acetaminophen and its glutathione, cysteine and N-acetylcysteine conjugates.

Sampling, sample preparation and analysis Cucumber seedlings treated with or without 5 mg L−1 acetaminophen were sacrificed at 0, 12, 24, 48, 72, 96, 120 and 144 h. Before sample preparation, roots were rinsed thoroughly with tap water. Harvested tissues were separated into roots and shoots and then frozen in liquid nitrogen. Enzyme activities of GST, glutathione reductase , γ-glutamylcysteine synthetase , and cytochrome P450, as well as contents of reduced glutathione in both roots and shoots, and lipid peroxidation were immediately analyzed after sampling, as described below. The remaining plant samples were freeze-dried and stored at −80 °C until chemical analysis. At each sampling time, nutrient solution samples and the aqueous root exudate samples were collected and mixed with equal volumes of methanol. The mixture was added with d4-acetaminophen as a surrogate, filtered through a PTFE syringe filter and stored at −20 °C before analysis. To quantify acetaminophen and its glutathione,indoor garden cysteine and Nacetylcysteine conjugates, the freeze-dried plant tissue samples were ground to a fine powder with a mortar and pestle, and a 0.5-g aliquot was used for solvent extraction. Each sample was spiked with d4- acetaminophen as the recovery surrogate, and extracted, sequentially, with 20 mL methyl tert-butyl ether , acetonitrile, and 0.5 mM HCl solution in an ultrasonic water bath for 20 min for each extraction. The supernatant from MTBE and acetonitrile extraction was combined after centrifugation and reconstituted in 1.0 mL methanol after drying under nitrogen . The extract was pooled with the supernatant from the HCl extraction. The pooled extract was loaded onto an Oasis™ HLB cartridge that was preconditioned with 6 mL methanol and 12 mL deionized water, and eluted with 15 mL methanol under gravity. The eluate was evaporated to dryness under N2, and the residue was recovered in 1.5 mL methanol:water mixture and filtered before analysis. The instrumental analysis was performed on a Waters ACQUITY ultra-performance liquid chromatography coupled to a Waters Micromass Triple Quadrupole mass spectrometer equipped with an electrospray ionization interface . Acetaminophen, and its glutathione, cysteine and N-acetylcysteine conjugates were separated using ACQUITY UPLC BEH C18 column with water and methanol as mobile phases. Electrospray ionization was operated in the positive mode. Detailed information on the instrumental analysis is given in Table S4.Fresh cucumber roots were frozen in liquid nitrogen, and homogenized on ice with 5 mL of 50 mM potassium phosphate buffer containing 1 mM EDTA and 1% polyvinylpyrrolidone. After centrifugation at 15,000 g at 4 °C for 20 min, the supernatant was used for the assay of activities of glutathione S-transferase, glutathione reductase, and cytochrome P450 . Measurement of GST activity was carried out spectrophotometrically after the glutathione and 1-chloro-2,4-dinitrobenzene adduct synthesis reaction . Briefly, GST activity was measured in 2 mL of a reaction mixture containing 50 mM PBS , 1 mM CDNB, 5 mM GSH and 100 μL enzyme extract. The increase in absorbance as a result of GSH-CDNB synthesis was determined at 340 nm for 5 min on a Cary 50 UV– Visible spectrophotometer . The GST activity was calculated using the extinction coefficient of GSH-CDNB .

To determine the activity of GR , a 200 μL aliquot of enzyme extract was added to the reaction mixture of 0.15 mM NADPH and 0.5 mM oxidized glutathione in 50 mM PBS . The absorbance decrease was monitored at 340 nm for 3 min after GSSG-dependent consumption of NADPH . For the assay of γ-ECS , plant tissues were homogenized and suspended in 5 mL of 0.1 M HCl. After centrifugation at 20,000 g for10 min at 4 °C, the supernatant was collected. The γ-ECS activity was measured according to Shan and Liang . An incubation solution was prepared with 800 μL 50 mM TrisHCl containing 0.25 mM glutamate, 10 mM ATP, 1 mM dithioerythritol, 2 mM cysteine, and 400 μL of the γ-ECS sample. The mixed solution was incubated for 1 h at 25 °C, and then 800 μL phosphorus agent was added and mixed. After incubation at 45 °C for 25 min, the absorbance at 660 nm was measured on the UV–Visible spectrophotometer. One unit of γ-ECS activity was defined as 1 mmol cysteine-dependently generated PO4 3− per minute. The levels of proteins in the enzyme extracts were determined using Coomassie Brilliant Blue G-250 . Brieflfly, 100 μL of enzyme extract was added to 5 mL of 0.01% Coomassie Brilliant Blue G- 250. After 5 min, the absorbance at 595 nm was measured on the UV– Visible spectrophotometer. The protein content of enzyme extracts was calculated by comparison with a standard curve using bovine serum albumin as the standard. The protein concentrations in the extracts were estimated using the BSA standard curve. The content of GSH was determined spectrophotometrically according to Jiang et al. . Plant tissues were homogenized in 5 mL of cold 5% meta-phosphoric acid on ice and centrifuged at 12,000 g at 4 °C for 15 min, and the supernatant was analyzed for GSH. To 0.5 mL of supernatant, 0.5 mL PBS and 0.5 mL of 5′5′-dithiobis-2- nitrobenzonic acid were added. After thorough mixing and incubation for 5 min, the absorbance at 412 nm was measured . Membrane lipid peroxidation, a typical indicator of stress phytotoxicity , was estimated by measuring the concentration of malondialdehyde, according to the reaction with thiobarbituric acid as described in Sun et al. .All treatments were performed in triplicates. A procedural blank and a sample duplicate were included in every batch of 10 samples to monitor background contamination and reproducibility, and the calculated relative standard deviations were b10%. The d4-Acetaminophen was used to account for any loss during sample preparation, matrixinduced ionization effects, and variations in instrumental response. Authentic standards were used to confirm the target analytes. A six-point calibration line was used for quantification with the r 2 values of at least 0.99 for all analytes . No acetaminophen or any of the conjugates was detected in the solvent or treatment blanks. Limits of detection and quantification for individual acetaminophen and the conjugates were calculated as 3 and 10 times the signal-to noise level from the low-level spiked samples . The recoveries in all samples were within acceptable limits ranging from 75 to 110%, demonstrating good method accuracy and precision. Extraction efficiency was N91% for acetaminophen and its conjugates. The plant position was regularly rearranged every 2 days to avoid side-effects.The level of acetaminophen in the nutrient solution without plant, after the removal of plant , or with cucumber plant, all decreased over time . Significant losses occurred in the unplanted control or the root exudate control . Considering that the cultivation system was not a sterile environment, both microbial and abiotic degradation may have contributed to the apparent acetaminophen dissipation in the solutions. This observation was consistent with Bartha et al. , who also reported the occurrence of plant-independent, biotic processes that decreased the available acetaminophen in plant solution.

Plant health and development are influenced by a broad range of microorganisms inhabiting the root rhizosphere, including beneficial associative bacteria. These bacteria are usually minor components of the rhizosphere microbial community but have been shown to significantly enhance plant growth and yield . This ability to enhance plant growth has been attributed to diverse mechanisms, including biological nitrogen fixation, production of phytohormones, enhancement of nutrient uptake , and bio-control of pathogens and pests . Plant growth-promoting bacteria can colonize roots either on the root surface or as endophytes without eliciting a noticeable plant defense response. Unlike some other bacterium-plant interactions that show strict host specificity, many PGPB can colonize a wide variety of plant species, including agriculturally important members of the Poaceae family, such as rice, maize, wheat, and a variety of bio-energy grasses . PGPB strains have been described for several host plants, including Setaria viridis, a model C4 plant, which is a close relative to a variety of bio-energy grasses. In a previous study, we demonstrated that S. viridis under lab conditions can obtain up to 100% of its nitrogen needs through biological nitrogen fixation mediated by diazotrophic PGPB . Among the strains used were the betaproteobacteria Azoarcus olearius and Herbaspirillum seropedicae, which exhibit strong growth-promoting ability . Azoarcus olearius DQS4T was originally isolated from oil-contaminated soil and, based on its genome sequence, this strain shows high similarity with the well-studied A. olearius strain BH72 . Strain DQS4T can colonize the roots of rice and Setaria to high levels and increase below- and above-ground biomass . H. seropedicae SmR1 is a well-studied endophytic bacterium that colonizes several plants, including maize, wheat, and Setaria. Recently,container size for strawberries in situ metabolomic profiling of S. viridis roots colonized by SmR1 demonstrated that inoculation induced a wide variety of plant metabolic changes, including those affecting nitrogen and phytohormone levels .

Thus, similar to many other, better-studied plant-microbe associations, PGPB inoculation appears to profoundly affect the metabolism of its host. To identify specific PGPB genes that contribute to plant root colonization, we applied transposon mutagenesis sequencing coupled with random barcoding. This technique assesses gene functionality in mutant strains through the generation of reusable libraries of unique and mapped mutant insertions.TnSeq is a powerful and sensitive approach to identify bacterial gene functions that play roles in bacterial fitness under researcher-defined growth conditions . Starting with information on the location and frequency of each mutation in the population, the change in mutational frequency after passage of the population through an environmental challenge leads to the identification of genes that are either essential, important, or detrimental to growth under that condition. For example, TnSeq of Streptococcus pneumoniae identified genes essential for bacterial basal growth, as well as genes involved in transcriptional regulation and carbohydrate transport . In the PGPB Pseudomonas simiae, TnSeq revealed genes involved in carbon metabolism and motility that could enhance or suppress colonization of Arabidopsis roots . In the present work, we applied standard TnSeq and a variant of TnSeq that uses random DNA barcodes to measure strain abundance to construct mutant libraries of strains DQS4T and SmR1, respectively, and to then evaluate genetic contributions to fitness on the host plant Setaria viridis.Gene essentiality is defined as its importance in maintaining competitive fitness under a condition of interest . For this study, two PGPB bacterial species were used, Azoarcus olearius DQS4T and Herbaspirillum seropedicae SmR1, both diazotrophic and capable of promoting plant growth . The mutagenized pool of each strain was inoculated onto Setaria viridis roots or a carbon augmented soil control with no plants.

Root-associated bacterial cells were recovered, transposon DNA was sequenced, and resulting reads were mapped to each bacterial genome. The relative incidence of a specific mutation was then determined and used to calculate fitness values. By high throughput sequence analysis of insertion mutants, we covered about 3,692 genes of DQS4T and 3,878 genes of SmR1 distributed throughout either strain’s genome. Thus, regardless of method, very good coverage of each genome was achieved. In the case of DQS4T transposon mutagenesis, 89.2% of 4,135 total genes were mapped to a gene harboring at least 1 insertion event. For the SmR1 library, 81.1% of the 4781 genes had identified mutations. We assume that genes without an insertion in the library likely identify those essential for bacterial growth in culture. Our screening identified 89 and 130 gene mutations that significantly affected the ability of strains DQS4T and SmR1 to colonize S. viridis roots, respectively. Given that our interest was to identify genes that exclusively affected root colonization, we excluded from further consideration any genes that also affected fitness under soil conditions . The genes affecting root colonization were categorized based on the phenotypes as either enhanced fitness value, mutations that increased root association ; or decreased fitness value, mutations that impaired root association . For the complete list of gene mutations affecting fitness under each condition, see Table S2. Unsurprisingly, many of the genes identified lacked a clear functional annotation, especially for A. olearius DQS4T . Even though each strain showed a distinct pattern of mutations that affected fitness, a few common gene functions were identified impairing root colonization in both strains. The most common COG categories important for SmR1 root colonization were those involved in amino acid transport and metabolism, followed by energy production and conversion and coenzyme transport and metabolism. In the case of DQS4Tthe most common COGs were cell wall/membrane/envelope bio-genesis and signal transduction, followed by translation, ribosomal structure and bio-genesis, and inorganic ion transport and metabolism .

Although we cannot rule out that the different mutagenesis methods could contribute to the different fitness profiles, the overall mutational coverage of the genomes of both strains suggest that such effects are likely minor. Hence, the results seem to suggest that any given PGPB strain will have different major requirements for root colonization reflecting the specific metabolic needs of the bacterium.Gene mutations that positively impacted the ability of bacterial strains to colonize roots were defined as those with a fitness score $ 1. We identified 8 and 14 genes in this category in A. olearius and H. seropedicae, respectively . This list included the Azoarcus gene predicted to encode a pyrroloquinoline quinone -dependent alcohol dehydrogenase , which is involved in the oxidoreductase process of ADH. Based on sequence alignment with Azoarcus olearius BH72,container size for blueberries the DQS_RS19730 gene encodes an ExaA5 protein that was previously reported to be induced upon exposure to ethanol as a carbon source . In our study, we found that exaA5 plays an important role in root colonization. Disruption of this gene enhanced root colonization of S. viridis roots . However, the mutant was less competitive in root colonization than the wild type at 1 and 3 days after inoculation but seemed to recover to wild-type levels by 5 days after inoculation . An insertion mutation in the gene encoding a diguanylate cyclase significantly increased bacterial root colonization 3 and 5 days postinoculation and showed the same trend in competition with the wild type . Diguanylate cyclases with a GGDEF activesite motif produce cyclic di-GMP and play a major role in the transition between motile and sessile bacterial lifestyles . We also identified an iron regulator outer membrane protein, TonB , presumably required for Fe31uptake . This protein was reported to interact with ExbA and ExbB, forming a complex that regulates iron acquisition . In SmR1, 6 of the 14 gene mutations that increased root colonization are predicted to play a role in flagellar biosynthesis. FliF, FliG, FlhB, and FliQ are structural components of the flagellar basal body . MotA is complexed with MotB and functions as a proton channel for torque generation . Our testing showed that the absence of flagellar genes in the SmR1 strain conveyed an advantage for root colonization in S. viridis 10 days after inoculation. We also identified a poly-3-hydroxybutyrate -related gene, a poly-3-hydroxyalkanoate depolymerase PhaZ1 , that enhanced colonization ability when mutated . PhaZ1 is a PHB depolymerization enzyme required for the granule mobilization . Furthermore, mutations in the genes encoding transcriptional regulators and ABC transporters also resulted in increased root colonization by strain SmR1 .Mutant strains corresponding to 81 DQS4T and 114 SmR1 genes were identified as significantly reducing root colonization . Therefore, these gene functions are normally required for efficient colonization by wild-type bacteria. Within this group of mutations, we identified two genes present in both DQS4T and SmR1: cheY , which is related to chemotaxis, and ampD , which is involved in peptidogly can degradation. Chemotaxis is directly involved in modulating the movement of the flagellum in response to attractants, helping bacterial orientation and active motion for survival toward favorable conditions .

In addition, four other genes encoding transmembrane chemoreceptors, also known as methyl-accepting chemotaxis proteins , were identified in H. seropedicae. The protein CheA undergoes autophosphorylation induced by MCPs, and CheR and CheW acts to modulate the phosphorylation state of CheY . Mutations in these genes impaired the capacity of H. seropedicae to colonize roots, consistent with a previous report .Analysis of the DQS4T TnSeq data identified a gene predicted to encode a two-component sensor histidine kinase containing a HAMP domain involved in signal conversion between the transmembrane-sensing and kinase activity control . One assumes that this histidine kinase is reacting to an unknown factor in the rhizosphere that normally enhances root association in wild-type cells. We also observed genes involved in cell wall formation, such as murI , a glutamate racemase responsible for the conversion of L-glutamate in D-glutamate, and murA , a UDP-N-acetylglucosamine 1-carboxyvinyltransferase responsible for converting UDP-N-acetylglucosamine to UDP-N-acetylglucosamine enolpyruvate. A previous study of H. seropedicae reported the importance of the mur genes for maize root association and also showed that their expression was repressed in the presence of naringenin. In addition, a N-acetyl-anhydromuranmyl-L-alanine amidase AmpD, likely involved in the degradation of peptidoglycan by hydrolysis of muropeptides that can serve as signals for induction of b-lactamase , was identified as important for root colonization in both strains. We also identified a putative transcriptional regulator of catechol 2,3-dioxygenase, a flavonoidrelated compound that might be involved in the degradation of aromatic compounds important for plant-bacterial interactions . Among such compounds are flavonoids that can serve as chemoattractants for rhizobia in legumes . Flavonoids can also mediate PGPB colonization, as reported for H. seropedicae colonization of Arabidopsis and the colonization of rice by Serratia spp. . Interestingly, mutations in genes involved in PHB metabolism—including PhaP1 , a phasin family protein that affects PHB production, and PhbA1 , an acetyl-CoA acetyltransferase— decreased root colonization. The importance of PHB metabolism in bacteria during plant root colonization was reported previously in studies of Setaria-Herbaspirillum and legumerhizobium symbiosis .To more definitively test the importance of individual genes identified in our screen, we selected candidate genes and created insertion mutations in 15 separate genes. These genes were selected to cover a diversity of putative functions representing operons containing multiple genes, as well as single genes, including those associated with both positive and negative fitness scores . Using a gnotobiotic system, a competitive colonization assay was performed in which colonization of S. viridis roots by the individual mutants was measured, as well as their ability to compete for colonization when coinoculated with the corresponding wild-type strain. Since root colonization is a dynamic process and might change over time, we evaluated competition at three different time points. For A. olearius DQS4T , we selected six genes where mutations reduced colonization, specifically a transcriptional regulator, ybeZ, ketoacyl ACP synthase , peaF, pcm, and purU . Mutants defective in genes that resulted in reduced colonization in competition with the wild-type strain showed delayed colonization of Setaria roots. Mutation of the transcriptional regulator gene represents the most extreme case where colonization was completely blocked when inoculated singly onto Setaria roots . This gene encodes an AphS protein known to be involved in the phenol degradation pathway, where phenol is converted into catechol via catechol 2,3-dioxygenase . Analysis of A. olearius DQS4T and BH72 genomes showed two that is produced by the wild type that can compensate for its loss in the mutant. As for the other mutants that impaired A. olearius DQS4T colonization ybeZ, ketoacyl ACP synthase , peaF, pcm, and purU, all showed a significant reduction in colonization that was most pronounced 3 and 5 days after inoculation both when inoculated individually or in competition with the wild-type strain .

Although we have arbitrarily classified surfactants as either causing either a “bright” or “dark” halo in oil drops surrounding a surfactant source, it is most probable that there is a spectrum of contact angles for the oil droplet that is dictated by the expected range of change of the various tensions by various surfactants as discussed above. Similarly, while all of our obtained mutants displayed bright halos, there is a possibility that the contact angles of the oil droplets could be slightly different, especially near mutants unable to produce syringafactin. However, we have not yet found a reliable method for measuring the contact angles of the atomized oil droplets observed with our assay, and no obvious differences in droplet shape were detected during microscopic observation of the droplets. It is not clear if there is an invariant correlation between a surfactant’s hydrophilic-lipophilic balance and the shape it imparts to oil droplets on an agar surface. It is, however, tempting to speculate on the utility of this assay in predicting important characteristics of novel surfactants. HLB values are a scalar factor that reflects the degree to which a surfactant is hydrophilic or lipophilic, with a value of zero reflecting a completely lipophilic molecule, a value of 10 corresponding to a compound with equivalent hydrophobic and hydrophilic groups, and values over 10 for predominantly hydrophilic molecules.For example, common surfactants such as SDS and Tween 20 have high HLB values and are therefore best suited for emulsifying a hydrophobic substance into a water phase. On the other hand, surfactants such as Silwet® L-77 with HLB values near 10 are more suited for wetting, or spreading of a water phase over surfaces such as leaves. These surfactants with balanced water- and oil- loving groups can be very effective as spreading agents, capable of lowering the surface tension of water below 30 mN/m. Rhamnolipid, with a predicted HLB of 9.5,plastic pots for planting which can lower the surface tension of water to 28 mN/m, is a highly effective spreading agent involved in bacterial motility. 

Although there is no consensus on the HLB of surfactin, it is also capable of lowering the surface tension of water to 27 mN/m, suggestive that it may also have an HLB near 10. Surfactants like Silwet® L-77 which had lower HLB values conferred bright halos in our assay. The surfactants with HLB values over 13, which are most ideal for emulsification of oil into water, did not cause the oil droplets to bead, resulting in dark halos when tested by the atomized oil assay. It is interesting that none of the biosurfactants tested conferred dark halos, suggesting that their primary roles are not as emulsifiers.It is noteworthy that the measurements of biosurfactant production using the halo method were strongly correlated with the swarming capability in mutants of P. syringae strain B728a. This suggests that the area covered by surfactants at the air/water interface as measured by our assay reflects a similar distance where swarming movement of bacteria across an aqueous agar surface is facilitated. Moreover, it is significant that drop-collapse activity was not a good indicator of the swarming ability of a strain, which raises the question of what specific properties make a surfactant a good lubricant that facilitates bacterial motility. Because the drop-collapse assay only detects surfactants that are able to greatly lower the surface tension of water, this property appears unnecessary for functions such as swarming. In addition, use of the drop-collapse assay in biological screens may cause a wide array of biologically active surfactants to be overlooked. In view of that, it is interesting that a syringafactin mutant of P. syringae strain B728a appears to produce a second surfactant that can promote swarming but not cause a drop-collapse. This is in contrast to a syringafactin mutant in P. syringae strain DC3000 which does not appear to produce this second surfactant. It is also striking that no mutants were identified in strain B728a that exhibited a total absence of surfactant halo, pointing to differential regulation of syringafactin and the remaining expressed surfactant. 

Furthermore, the disruption of pmpR apparently causes the down-regulation of syringafactin while conferring up-regulation of the other surfactant, suggesting its role in regulating both surfactants. While both P. syringae strains are pathogenic to plants, strain B728a is a much better epiphyte than DC3000. Perhaps this second surfactant is particularly useful for the lifestyle of epiphytes such as strain B728a on waxy leaf surfaces. We are actively pursuing the identity and specific properties of this second surfactant. The phytotoxins syringomycin and syringopeptin have been suggested to possess surfactant activities , although preliminary results have not yet provided support for the identity of either of these surfactants as the second surfactant. It is possible that combining one of the mutations found from this screen with a syfA or syfB mutation could reveal the identity of the second surfactant Some, but not all of the genes found to regulate both biosurfactant production and swarming ability in P. syringae have homologs that influence swarming in Pseudomonas aeruginosa. Disruption of Psyr_3619, encoding an RNA helicase, conferred a similar reduction in swarming as that seen in blockage of its homolog PA2840 in P. aeruginosa. Likewise, disruption of pmpR in P. aeruginosa, a homolog of Psyr_1407, resulted in enhanced swarming in both species. It is significant that P. syringae B728a mutations were not identified in homologs of any of the many other genes found to alter swarming in P. aeruginosa despite the near completeness of the mutant library, emphasizing that the surfactants that contribute to swarming in these strains differ and/or that many factors other than biosurfactant production contribute to swarming ability. It is also noteworthy that relatively few different genes apparently contribute to biosurfactant production in P. syringae B728a. The disruptions of only 12 unique genes, identified from over 7,000 screened mutants,strawberries in a pot were found to alter biosurfactant production. Assuming random transposon insertion, we predict that we have screened a library of approximately 77% of the P. syringae B728a genes. Although we have identified many of the mutations which have an effect on measured surfactant halos, we may have missed a number of mutations which negatively affected syringafactin production but were masked by a compensatory increase in production of the second surfactant.

For life on the leaf surface, Pseudomonads have been shown to employ a variety of traits to grow and survive despite fluctuating water availability. In response to desiccation stress, Pseudomonads produce alginate in order to maintain a hydrated micro-environment. Our finding of multiple components of the AlgT regulatory pathway among mutants of strain B728a with altered biosurfactant production could suggest an intimate relationship between water availability and biosurfactant production. This potential relationship warrants further exploration of either the AlgT pathway or perhaps alginate production itself as a regulator of surfactant production. The role of biosurfactants on the leaf surface is most likely complex, and as such may likely prove to have very complex regulatory networks. The atomized oil assay has revealed a likely diversity of biosurfactants that are produced by strain B728a and their complex patterns of expression, details that would have been difficult to discern using other assays for biosurfactant production. The tools and genetic resources developed here should prove useful in further studies of the roles of surfactants in the interaction of P. syringae with plants. Biosurfactants, or biologically-produced surface active agents, have received wide attention mostly for their potential for hydrocarbon dispersion and remediation.However, a wide variety of roles for biosurfactants have been since described, from biofilm formation to inhibitory activity against pathogenic organisms, sparking a renewed interest in their discovery. Given this interest in biosurfactants, the lack of knowledge of the distribution and frequency of occurrence of surfactant production in the environment is remarkable. Comprehensive examinations of biosurfactant production are lacking, and studies that have addressed this trait in a given environment can seldom be compared with those of other habitats ; both the screening methods used, as well as pre-screening culturing conditions such as medium and incubation conditions usually vary widely between studies. In a recent report we described a high-throughput assay which utilizes the application of atomized oil droplets to rapidly detect biosurfactants produced by bacteria on the surface of agar plates. This method has advantages over other common assays such as droplet collapse assays in that it can be performed for many colonies simultaneously after limited growth, does not require sample preparation of culture supernatants, and thus is suited for high throughput screening for surfactant producing strains. Moreover, this method is capable of detecting much lower concentrations of surfactants than the drop collapse assay, and therefore in principle is capable of identifying biosurfactant producing strains that would escape detection with most other methods. However, since the atomized oil assay has not yet been tested on a broad range of environmental isolates, in this study we address whether the range of strains that it can detect includes all of those detectable by the drop collapse assay.

Furthermore, although the atomized oil assay has proven effective at detecting surfactants on agar plates, traditionally broth culture supernatants are screened for biosurfactant activity using the drop collapse assay. Depending on the properties of the surface-active compound and its biological role for the producing strain, its production may depend strongly on whether the producing cells are situated at a surface or not. Since a large difference in the transcriptomes of bacteria grown planktonically versus on surfaces have been described, with about one-third of genes differentially regulated , it seems likely that biosurfactant production itself may be strongly influenced by cell culture conditions. Surface sensing is an important cue for many species to transition to surface-associated behavior such as swarming, whereby cells move across a moist surface utilizing flagella and surfactant. Although the surface regulation of flagella has been well documented , the regulation of surfactant production by surfaces has not yet been explored and will be addressed in this report. Insight into the role of biosurfactants would benefit from a better understanding of the numerical distribution of surfactant producers in different environments. A variety of isolated reports have described collections of biosurfactant producers from aqueous environments, polluted/unpolluted soils, and even clouds, with estimates of their frequency in culturable bacterial communities ranging from less than 3 to as much as 50%, but typically around 10%. However, no encompassing model that describes the selection for such a trait has emerged from these studies, perhaps because few comparative analyses of habitats have been performed. We hypothesize that hydrophobic surfaces are habitats that would be particularly selective for bacteria that produce surface active compounds. The surface of leaves that are usually covered with wax would constitute such a habitat, although surfactant production in this habitat has seldom been investigated. In order to survive on leaf surfaces, epiphytes must be able to access limited and spatially heterogeneous nutrient supplies and endure daily fluctuations in moisture availability on a water-repellent surface. Epiphytic bacteria could potentially use biosurfactants to increase the wetability of the leaf, to enhance diffusion of nutrients across the waxy cuticle, and/or aid in motility to favorable growth sites. Despite the substantial potential role of biosurfactants on leaves, only a few studies have examined their production in the phyllosphere, all of which have focused on their possible ecological role in only specific strains and have not addressed the frequency of surfactant producers on leaf surfaces. A comprehensive examination of the phyllosphere inhabitants might reveal strains and biosurfactants not normally encountered in other habitats, and would address the hypothesis of surface enrichment of producing strains. In this study we compare the frequency of surfactant producers in the phyllosphere to those in soil and water environments. We compare the atomized oil assay with the drop collapse assay to characterize surfactants made by a collection of environmental strains, further demonstrating the usefulness of this assay in high-throughput screening and its much higher sensitivity for all types of biosurfactants encountered, many of which are hydrophobic and poorly detectable by the droplet collapse assay. We also investigate the influence of planktonic versus surface-associated culture conditions on the production of biosurfactants from our environmental isolates, and find evidence for frequent contact-dependent production of surface active compounds.

Worldwide use of neonicotinoids continues to expand as pest populations develop resistance to the once-widely-used pesticide classes including the organophosphates and methylcarbamates. The most commonly used neonicotinoids are IMI and TMX, the primary focus of these studies. The overall goal is to further understand the metabolism of neonicotinoids relative to: in vivo importance of AOX, metabolic pathways of the novel neonicotinoid, cycloxaprid and mechanisms of TMX hepatotoxicity and hepatocarcinogenicity. CYPs have been shown to be involved in in vitro and in vivo neonicotinoid metabolism, but the relative in vivo importance of AOX is unknown. AOX is implicated to play a role in the nitroreduction of N-nitroguanidine neonicotinoids, the most prominent subclass. There is considerable variability in the activity of AOX between species and individuals which may be reflected in differences in neonicotinoid metabolism and detoxification. Secondly, CYC is a new neonicotinoid that is under development to control IMI-resistant pests. However, its metabolic pathway has yet to be determined, particularly in reverting to its potent nAChR agonist precursor, nitromethylene-imidazole. Finally, TMX is the only neonicotinoid to produce liver toxicity and tumors in chronically-treated mice, but not rats. Earlier studies concluded that formation of dm TMX and iNOS inhibition by dm-CLO is likely the mechanism of TMX toxicity. Furthermore, differences in metabolic rates between species may explain the mouse specific toxicity. However, the molecular mechanism of TMX or dm-TMX hepatotoxicity/ hepatocarcinogenicity remains unclear. It is critical to fully understand the metabolic/ enzymatic pathways of neonicotinoids and mechanisms of toxicity as their use continues to increase and for future pesticide design.The nitro substituent on neonicotinoids is important relative to their potency and selectivity for the insect nAChR. From the seven commercial neonicotinoids, approximately 100 metabolites have been identified in plants and mammals, some of which are bio-activated and can interact with the mammalian nAChR. 

A number of studies have demonstrated the importance of CYPs in neonicotinoid metabolism in vitro and in vivo. However,macetas plastico cuadradas the role of AOX in neonicotinoid metabolism has yet to be established in vivo, especially in the oxidative- and CYP-rich environment of the liver. AOX is important in xenobiotic metabolism. This enzyme is expressed mainly in liver but is also present in many other tissues with variations in activity depending on species, gender, age, drug usage and disease states. Tungsten or hydralazine in the diet or drinking water results in reduced AOX activity in guinea pigs, rabbits and mice. There are even notable differences in AOX activity between strains of mice , e.g. compared to CD-1 mice, the DBA/2 strain is deficient in the expression of AOX homologue 1 and homologue 2 and has reduced expression of AOX1. Since AOH1 and AOX1 are the primary AOX genes expressed in mouse liver , DBA/2 mice are an appropriate AOX-deficient model for studies on in vivo mammalian xenobiotic metabolism. The wide range of inter- and intra-species AOX activity may result in different rates of neonicotinoid metabolism and detoxification in mammals and insects. Despite the increasing significance of AOX, there have been very few studies examining the in vivo contribution of this enzyme to xenobiotic metabolism. Mice can serve as a surrogate for humans since AOX activity in IMI nitroreduction in vitro is comparable between these two species. This study uses chemical inhibitors and genetic deficiency for mice and Drosophila melanogaster to evaluate the relevance of AOX in neonicotinoid metabolism in vivo. Mouse liver cytosol and microsomes were prepared by homogenizing liver in ice-cold PBS using a Sonic Dismembrator followed by centrifugation of the homogenate at 1,000g for 10 min and then the supernatant at 10,000g for 30 min. An aliquot of the 10,000g supernatant was recovered for AOX activity analysis and the remainder was centrifuged at 100,000g for 1 h to collect the CYP-containing microsomal pellet fraction which was resuspended in PBS for protein measurement and the CYP activity assay.

Mouse liver cytosol was added to 50 µM DMAC solution and the reaction monitored by an absorbance decrease using a VersaMax microplate reader at 398 nm for 5 min with an average control value of -18.4 mOD/min. 7-Ethoxycoumarin is a broad-specificity substrate used to measure the activity of many CYP enzymes by monitoring the oxidation to 7-hydroxycoumarin. Microsomes were mixed with 50 mM 7- ethoxycoumarin in assay buffer glycerol and 0.1 mM EDTA and prewarmed at 37°C for 5 min. After addition of 10 mM NADPH , reactions were incubated at 37°C for 30 min in a shaking water bath.Samples were extracted with chloroform , briefly vortexed, then centrifuged at 3,000g for 5 min. The organic phase was removed and added to 30 mM sodium borate and vortexed. Following centrifugation at 3,000g for 5 min, the upper layer was recovered and plated on a Costar 96-well black plate and fluorescence read at an excitation wavelength of 370 nm and an emission wavelength of 460 nm using a SpectraMax M2 Microplate Reader with an average control value of 11.2 nmol 7-hydroxycoumarin/mg protein.AOX is a potentially important factor in drug metabolism with many studies examining its in vitro inhibition and the proposed effects on xenobiotic action. There is a wide range of AOX activity between species with rabbits, monkeys and humans the highest, mice intermediate and rats and dogs having the lowest activity. This same species dependent relationship is also observed for in vitro IMI nitroreduction by liver cytosol. Tungsten and hydralazine treatments provide a way to reduce AOX activity in vivo in mammals to evaluate its relevance in xenobiotic metabolism. Tungsten replaces molybdenum at the active center of AOX, rendering it inactive , but the mechanism of AOX inactivation by hydralazine is unknown.The level of AOX inhibition by tungsten treatment in mice was less than that by hydralazine , a difference reflected in their effect on IMI metabolism. Hydralazine treatment resulted in significantly reduced IMI metabolism to IMI-NNO and IMI-NH,maceta redonda but tungsten treatment only significantly reduced IMI metabolism to IMI-NH. There are four AOX genes in mice with two of the variants being expressed in the liver, AOH1 and AOX1. DBA/2 mice are completely deficient in the expression of AOH1 and have low expression of AOX1 compared to CD-1 mice. Our data also establish that DBA/2 mice have significantly lower AOX activity in the liver and further show that the reduced AOX activity decreased IMI metabolism to IMI-NNO and IMI-NH, but not to IMI-5-OH or IMI-ole.

The AOX-generated IMI metabolites are not all detoxification products. IMI-NH is a likely contributor to the nicotinic effects of IMI. It is over 300 times more potent than IMI at the mammalian nAChR and the mouse ip toxicity is also increased several fold. IMI-NNO retains insecticidal activity and as an N-nitroso compound it was subjected to extensive toxicological tests and cleared of potential problems. Our study concludes that reduced AOX activity is tightly correlated with reduced IMI metabolism to IMI-NNO and IMI-NH indicating that these products are mostly from AOX, not CYPs. Based on the metabolic sequence and relevant correlations, IMI-NH is mostly formed via IMI-NNO rather than another pathway. This is the first report on the metabolism of CYC with an emphasis on in vivo metabolites in mice at 15 and 120 min post-treatment. Metabolites are not conclusively identified since synthetic standards were not available. However, based on calculated m/z values and characteristic chlorine isotope patterns, it is concluded that CYC is converted in part to NMI, but is mostly oxidized to multiple mono- and dihydroxylation products within 15 min and dissipate by 120 min in liver. There are many possible isomeric CYC oxidation products from hydroxylation on the 6-, 7-, 10- or 11- position in each case with two possible stereoisomers of which five are detected as distinct peaks by LC/MS. Minor products include 2-CYC and nitroreduction on the nitro group to NO-CYC and NH2-CYC. CYC is readily hydrolyzed to NMI. Therefore, extraction and analytical conditions were carefully chosen to limit degradation. However, minor amounts of NMI detected may be due to degradation of CYC rather than its in vivo metabolism. NMI per se was not extensively metabolized. Only minor amounts of one hydroxylation and one nitroso product were evident. Detection methods may limit observing the extent of metabolism since only 3 mg/kg was administered to mice. The findings reported here lay the background for future studies on characterization of metabolites and in vitro species comparisons. In vitro conditions will allow detection of more metabolites and the enzymes responsible for their formation can be determined. 

TMX undergoes metabolic activation to CLO in insects, plants and mice. The neonicotinoids generally have favorable mammalian toxicology with the exception of TMX which is a hepatotoxicant and hepatocarcinogen in mice but not rats or dogs. The mechanism of this TMX- and mouse-specific hepatotoxicity/ hepatocarcinogenicity is of considerable interest relative to neonicotinoid risk assessment. Green et al.observed that liver microsomal metabolic rates are greater for mouse than rat or human in the production of TMX metabolites, dm-TMX, CLO and dm-CLO. They also found that mouse-specific adverse effects of TMX are due to dm-TMX exacerbated by dm-CLO which mimics the structure of L-NAME, a standard inhibitor of iNOS, an enzyme with a regulatory role in the development of hepatotoxicity. The structure-activity relationships of neonicotinoids as hepatotoxicants or hepatocarcinogens help focus mechanistic hypotheses on specific molecular substituents. TMX and dm-TMX are hepatotoxicants/ hepatocarcinogens and contain the oxadiazinane substituent uniquely among the neonicotinoids so this moiety is of particular interest. N-Methyl substituents on five of the neonicotinoids are not the hepatotoxic moiety because dm-TMX lacks this group. The Green et al.hypothesis is that dm-TMX is the hepatotoxicant exacerbated by dm-CLO as an iNOS inhibitor. The present study examines an alternative hypothesis that the unique aspect of the oxadiazinane moiety is its metabolic conversion to HCHO and N-methylol intermediates , the ultimate hepatotoxicants and hepatocarcinogens which may be synergized by dm-CLO as a NOS inhibitor.high-throughput oxymyoglobin assay by following the procedure of Dawson and Knowles. To determine nNOS inhibition, HEK293 cells overexpressing rat nNOS were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 0.4 mg/mL geneticin. For enzyme activity assays, cells were cultured in Costar 96-well black plates and treated with 5 µM A23187 ionophore to activate nNOS then with dm-CLO or L-NAME for 8 h. To measure nitric oxide production, cell media was removed and replaced with reaction buffer containing 1 mM L-arginine, 10 µM 4,5-diaminofluorescein diacetate , 1 mM NADPH and dm-CLO or L-NAME. After a 2-h incubation in the dark, fluorescence was read at an excitation wavelength of 490 nm and an emission wavelength of 520 nm using a SpectraMax M2 Microplate Reader.Mouse liver microsomes were prepared by homogenizing livers from male albino Swiss Webster mice in PBS followed by differential centrifugation of the supernatant. The microsomal 100,000g pellet was resuspended in PBS and protein concentration measured. Liver microsomes for species comparison studies were from BD Biosciences. Recombinant CYP3A4 was compared to recombinant CYP2C19 , the two isoforms previously shown to be responsible for TMX metabolism. Each neonicotinoid was incubated with microsomes from mouse, rat or human or rCYP isoform and 0 or 1 mM NADPH in PBS for 1 h at 37°C. Alachlor and hexamethylphosphoramide , compounds known to produce HCHO via N-methylol intermediates on activation by CYPs , were also incubated under the same conditions with mouse liver microsomes alone or with NADPH. Enzymatic reactions were terminated by addition of 25% ZnSO4 aqueous solution and saturated Ba2 aqueous solution. Samples were briefly vortexed and placed on ice for 5 min then centrifuged at 18,000g for 5 min. HCHO levels were analyzed after conversion to the 2,4-dinitrophenylhydrazine derivative. An aliquot of the supernatant was mixed with 7.2 mM DNPH and incubated at room temperature for 30 min followed by addition of carbon tetrachloride , vortexing for 30 seconds and a final incubation at room temperature for 30 min. The lower organic layer was evaporated to dryness under N2 at 25°C and resuspended in 80:20:0.1 ACN/water/HCO2H and filtered through 0.2 µm nylon for HPLC analysis. Samples were analyzed on a Waters Alliance 2695 HPLC equipped with an Agilent Zorbax SB-C18 column and Waters Alliance 2487 dual UV absorbance detector.

A constraint in one system could not only affect economic security in that system but could inhibit access in another. Therefore, the nexus provides a powerful means to improve synergies in food, energy, and water production , to identify how stressing food, energy and/or water systems creates resource vulnerabilities and/or resource scarcities in all three, to understand and quantify the production of ecosystem services, and to develop climate adaptation strategies. However, historically, food, energy, and water systems have been pigeonholed politically as well as broken up into small disjointed pieces that cross political boundaries and do not align with bio-regions or watersheds. This type of disjointed management leaves policy makers ill-equipped to provide resilient management strategies. Thus, the success of using the nexus concept to improve food, energy, and water systems will likely depend on how it incorporates issues surrounding resource governance , including how governance and the discourse of securitization become a way to legitimize political agendas. Many have argued that the nexus discourse of security places economic variables over access to resources for the world’s poor, an idea that can be traced back to Foucault’s theory of the linkages between security and the circulation of the global economy. In addition to this discursive ‘securitization’, resource governance outcomes are based on the larger political goals of the government or states involved in policy making. The connections between discourse, policy,diametro maceta 10 litros and land management, therefore, raise important questions regarding how policy is already impacting nexus outcomes and communities on the ground.

Given the recent emergence of the FEW concept, few studies to date have explored the use of the nexus concept simultaneously with an analysis of governance structures. To address this gap, we present a quantitative application of the FEW nexus concept to study resource vulnerabilities and scarcities in the Lower Colorado River Basin in California, Arizona, and Nevada, U.S.A. We analyze the nexus within its sociopolitical, economic, and bio-regional context that determine what resources are available, used, produced, and traded. We take a case study approach, as case studies are best able to translate the on-the-ground nexus realities of a variety of institutions, bureaucracies, and stakeholders across space, time, and scale. Our goal was to understand how the governance structure of the Colorado River constrains the utility of the nexus approach to deal with future stresses. To do this, we first quantified the nexus by identifying the local and global linkages between food, energy, and water as well as the choices confronting water managers, and the Indian Reservations in the study area. We use these findings to look for the emergence of tipping points under two different scenarios: drought and increased demand for alfalfa. We then discuss how the very rigid water laws in the LCRB constrain the ability to improve resource management and respond to these tipping points using nexus thinking. While the main focus of this paper is on how economic and hydraulic factors influence FEW’s nexus governance, in the discussion we also examine the impact of institutional and political factors as well as geopolitics across the transnational boundary between the U.S. and Mexico.The Colorado River Basin has a semiarid to arid climate with an average of 40 cm annual precipitation that originates as snow pack in the Rocky Mountains and contributes to about 70% of the total stream flow in the basin. However, temperatures have been rising for the past century, with winter temperatures increasing more than summer temperatures on average by 2 °C. 

Temperatures are predicted to rise at least another 1.1 to 2.0 °C by 2050. These higher temperatures increase evaporation rates and have coincided with a reduction in snow pack and snow melt in the UCRB. In addition, the LCRB has been in a drought since 2002, though at the same time the UCRB has experienced less severe or no drought conditions, outside of extremely dry periods in 2003 to 2004, and 2012. Historically, paleo climatic records indicate pervasive and long-lasting periods of drought occur in the region. However, more recently, the area has received significantly more snowfall.Its water elevation has been decreasing since 2000, which can be seen visually in aerial imagery.The Colorado River is operated under the “Law of the River,” which is comprised of a variety of U.S. federal laws, agreements, court decisions, and regulatory guidelines. These laws apply to seven Western U.S. states: Arizona, California, Colorado, Nevada, New Mexico, Utah, and Wyoming, and Mexico that utilize water from the river. The Law of the River allocates to each basin 7.5 million acre-feet 1 of water per year in perpetuity, with some exceptions that account for water scarcity. High variability in precipitation in the Rocky Mountains results in 5 MAF to 25 MAF of flow in any given year with an average annual flow of less than 16.4 MAF/yr. In terms of usage, on average 91.4% of the allocated water is used by municipalities, for power, or agriculture. The UCRB Indian reservations receive 1 MAF a year, while the LCRB Native American reservations are allotted 0.9 MAF a year. While this water is split amongst the 20 reservations in the LCRB, only six reservations have had their water rights quantified. This has resulted in a disproportional amount of water allotted per person on these reservations compared with the rest of the basin. Specifically, 6.7 AC-FT per person per year is allotted to Lower Basin Indian reservations while 0.9 AC-FT per person per year is allotted to the rest of the LCRB. It should be noted that the water allotments of both basins , Mexico ,macetas cultivo and the Native American Reservations , adds up to 18.4 MAF, 2 MAF more than the yearly average stream flow of the Colorado River not accounting for climatic fluctuations.

The rigid allotments based on the Law of the River have also resulted in most of the agriculture production in the study area taking place on Indian reservations. Prior to the Law of the River, American Indians practiced flood farming practices for thousands of years based on characteristic seasonal rains , as well as dry farming. Today, however, practices are much more water-intensive on Indian Reservations.The Colorado river allows local economies to exist in a semi-arid environment with two-thirds of Arizona’s, California’s, and Nevada’s state gross products dependent on the Lower Colorado River. The river provides 657.5 billion dollars of direct, indirect, and induced GDP to California; 185 billion dollars to Arizona; and 115.4 billion dollars to Nevada. Focusing on food systems, agricultural production in the study area is largely situated on Indian reservations due to their high allotment of river water.For alfalfa, one of the most common crops in the region, the water that is used to flood the fields is sent back into the canals. The high-water availability along with the warm climate allows the production of twelve harvests of alfalfa each year. Most of the alfalfa is exported as fodder to China, followed by Japan, Mexico, South Korea, and the EU.A diversity of data sources drawn from the Food and Agriculture Organization of the United Nations , the United States Department of Agriculture , and the Energy Information Association were used to quantify the connections between food, energy and water.Energy was quantified in terms of net generation and cost for hydroelectric, natural gas, solar, and wind generated in the region for 2001 to 2016. Food production was quantified in terms of area of agricultural crops as well as the price that farmers spent on production for 2008 to 2015. Only crops that took up greater than 1% of total area were analyzed. To determine the total impact of one sector on another , we combined the total amount of water used in energy or food, and the total amount of energy used to pump water or produce food.Water-energy linkages were quantified in two different ways. First, the amount of water used in energy production 2001 to 2016 was calculated by multiplying the appropriate water consumption factors of electricity generated from natural gas from Spang et al., 2014 by the amount of energy produced monthly from 2001 to 2016. Water used in natural gas extraction for gas used in the study area was not included since there are no natural gas extraction operations within the basin. Hydroelectric projects have no net water consumption, but reservoirs make evaporation rates higher, especially with higher temperatures. Second, energy used to transport water in the Colorado River Aqueduct was quantified using data obtained from the Metropolitan Water District of Southern California for 2001 to 2016.

The aqueduct conveys water from Lake Havasu over the Santa Ana Mountains and to cities in Southern California, including Los Angeles. In addition, energy used to transport water from Lake Havasu to Phoenix and Tucson via the Central Arizona Project was drawn from a previous study by Kleiman. A final water-energy linkage, the amount of energy need to purify wastewater for domestic consumption was not quantified due to lack of data. Generally, however, wastewater treatment accounts for ∼3% of energy used in the United States.First, information on specific crop water usage was estimated using evapotranspiration rates in ft/growing period and average area cultivated annually for six of the seven most common monocrops in the LCRB. All data came from FAO estimates except for lettuce. FAO evapotranspiration estimates are given in mm/growing period, however, we converted the units to ft/growing period so we could easily translate the measurement to volume of water in AC-FT. Second, data for 2007, 2012, and 2013 from the National Agricultural Statistics Service for the entire Lower Colorado River Basin was used to estimate yearly water used for irrigation. In order to calculate the data at the watershed subunit level, we used the United States Agriculture Service 2012 Census and Cropland Data Layer to estimate the percent of irrigated land out of the total acres of cropland. The proportion of cropland that was irrigated varied based on data year, in 2013, 67.74% of cropland was irrigated; in 2012, 75.82% of cropland was irrigated; and in 2007, 71.21% cropland was irrigated. We averaged the percent of cropland irrigated in the watershed subunit for 2007, 2012, 2013 to extrapolate to the remaining years from 2008 to 2015. The average irrigation estimate was used in the production scenarios to understand responsiveness of irrigation and crop production to changes in climate.The linkage between food and energy was quantified in two ways. First, we estimated the amount of direct usage of fossil fuels by calculating the amount of fuel farmers purchased to run farm machinery in 2012. Second, using the 2013 NASS estimation for energy expended in irrigation for the entire LCRB, estimated at $49.00 per acre, we estimated the cost for irrigation in the study area. Using the cost per amount of energy we converted $49.00 per acre to the amount of energy in MWH used for irrigation in the study area. Assuming that the price of energy needed to irrigate remained constant, we calculated the energy expense for all years using the estimated irrigated cropland and cost per amount of energy for those years.As expected for a temperate semi-arid climate, the amount of water consumed followed a seasonal pattern with increased use during the summer months and decreased use during winter. Across Arizona, California, and Nevada, the main consumptive water use was either irrigation or municipal use, depending on the year. In 2013, the biggest consumers in Arizona and California were large cities such as Phoenix, Tucson, and irrigation districts. Water to cities in Arizona was transferred through the Central Arizona Project with over 1.5 MAF consumed. The Imperial Irrigation District was California’s largest water consumer in 2013, consuming more than 2.5 MAF of water. The Metropolitan Water District that transfers water to Los Angeles consumed less than the Central Arizona Project with just over one MAF. In Nevada, there are no consumptive uses over 100,000ACFT. However, the City of Las Vegas receives its water right before it reaches Lake Mead, so this is not accounted for in our analysis.

In multiple studies, slow sand filtration has been useful to remove and eliminate propagules of plant pathogens: many species of phytophthora – P. cinnamomi , P. cryptogea , P. nicotianae , and species of Pythium , Fusarium , E. coli , and nematodes including Radopholus similis. These zoosporic fungi, viruses, and nematodes cause crop damage including severe root rot and if not treated in a recirculating nursery and greenhouse system, widespread epidemic may impact all the crops within the facilities. While SSF has been shown to remove plant pathogens, its efficacy to remove other compounds has yet to be explored in depth. Contaminants of emerging concern , also named “emerging contaminants”, are a group of contaminants that consist of pharmaceutically active compounds, endocrine disrupting compounds, personal care products, plasticizers, pesticides and herbicides, and flame retardants that are found in trace amounts in the environments, primarily discharged from wastewater treatment plants that insufficiently treat these contaminants through secondary and tertiary treatment. Non-point sources, such as overland flow during rainfall or land drainage in agricultural areas deliver veterinary medicines and pesticide runoff to surface water or groundwater. The existence of emerging contaminants is unknown in the environment due to the lack of monitoring and due to their low concentrations in surface waters. Many of these compounds vary due to their application and consumption from region to region and will depend on the efficiency of removal by wastewater treatment plants. Technologies already proven effective in removing contaminants are activated carbon ,maceta 25l ozonation and advanced oxidation processes , and membrane filtration. However, these processes are costly and require large amounts of resources to operate.

In turn, bio-filtration systems are simple to operate, relatively low in cost and maintenance, removes both turbidity and propagules of pathogens, and overall improves the quality of the water. Thus, bio-filtration like SSF may offer a low cost alternative for the treatment of contaminants in wastewater.The most commonly researched bio-filtration systems to treat emerging contaminants are managed aquifer recharge processes. Managed aquifer recharge processes are robust and cost-effective systems and include a variety of applications such as aquifer storage and recovery, infiltration ponds, percolation tanks, soil aquifer treatment, and sand dams. MAR systems like bank filtration and artificial recharge are adopted by wastewater treatment plants to reduce the cost of using more costly advanced treatment systems like nanofiltration and reverse osmosis. MAR systems are typically adopted if surface water quality is inadequate or if the amount of raw water like in groundwater is not sufficient ; MAR systems can replenish these natural systems. However, in comparison to SSF, biodegradation in MAR systems are very important mechanisms, much more than sorption as sorption sites can become exhausted or desportion can occur. This practice may not be as easy to maintain like slow sand filters, as sand can be back washed and the supernatant of the disturbed schmutzdecke can be drained. Also, it is suggested that MAR such as bank filtration may not be optimal for smaller operating facilities that have less land space. There have been many studies that focus on biofilm reactors such as bank filtration and underground dams that can remove organic micropollutants but these are not usually designed, only grown in natural conditions. Therefore these systems are very limited and may need to overcome obstacles of releasing WWTP effluent water directly to natural waters. However, given substantial research on MAR systems, pharmaceuticals and endocrine disrupting compounds, which are usually main contaminants of interest due to their potentially adverse effects on human health and aquatic life even at low concentrations , have shown positive results for their removal.

Most of these studies were laboratory scaled and field studies. Some contaminants researched in MAR systems are endocrine disrupting compounds , antiseptics , pharmaceuticals , and disinfection by-products. Baumgarten et al. investigated the removal of poorly degradable antibiotic sulfamethoxazole in laboratory columns for its removal in bank filtration. Results showed that 60% of sulfamethoxazole was removed within 14 days of column passage in aerobic conditions while no removal occurred under anoxic conditions in a 2 year long system operation. The degradation of poorly degradable compounds in aerobic conditions may give bank filtration its benefits in the top layers of sand, but shows complications with anoxic conditions, which can be representative of some MAR and SSF. Adaptation of the system may require long operation time as would be realistic in an actual bank filtration site. Thus, more biofilter systems with MAR and SSF should be studied for their processes that provide both oxic and anoxic conditions. Typically, varying redox conditions are effective for removing redox-sensitive organic micropollutants during MAR. Maeng et al. summarized the literature in a review regarding removal efficiencies of CECs using bank filtration and aquifer recharge column and full scale studies. To briefly summarize that work, there are CECs that are promising for removal by filtration and others which are more recalcitrant such as carbamazepine are ineffective. Antibiotics in a study by Heberer et al. investigated 19 targeted antibiotics at a lake bank filtration site in Berlin, Germany for 2.5 years. They detected 7 out of 19 target antibiotics. All antibiotics were completely removed after 2-4 months of travel time except for sulfamethoxazole, which in previous studies was discovered to be redox-dependent that degrades more effectively in anoxic conditions. Depending on the residence time, removal of antibiotics can increase. It can be concluded MAR is an effective treatment step for removing antibiotics, giving way for other filtration studies to filter veterinary antibiotics that may potentially be found in runoff from dairy farms. For non-steroidal anti-inflammatory drugs and analgesics, many have been removed at rates greater than 50% during bank filtration and aquifer recharge systems.

Field and laboratory scale studies have shown significant removals of diclofenac, ibuprofen, naproxen, and phenazone during soil passage. Diclofenac, ibuprofen, and naproxen have moderately high octanol-water partition coefficients , suggesting sorption would be the main mechanism of removal. Phenazone, however,frambuesas en macetas is more redox-dependent and can be removed under oxic conditions than anoxic conditions. Therefore, it is necessary to monitor the pH during soil passage as these NSAIDs may remain as ionic species in the aquatic environment, with more potential to be sorbed. Anticonvulsant pharmaceuticals have been shown numerous times their persistency in degradation in multiple treatment methods. Carbamazepine is one of the notorious poorly degradable compounds and has low removal in wastewater treatment plants. Drewes et al. showed no change in carbamazepine and primidone concentrations in soil aquifer treatment for estimated travel times up to six years. The extended research on this contaminant concluded bank filtration and aquifer recharge are not effective for anticonvulsant removal. Antidepressants removal has still yet to be studied. A study by Snyder et al. investigated three antidepressants and their fate during a pilot scale bank filtration. Fluoxetine was removed significantly at 99% and meprobamate was only 66%. There could be more research developed in understanding the fate of more antidepressants since they are commonly used drugs in the pharmaceutical industry. Lipid regulators are similar to NSAIDs, where they remain in ionic species. Thus, pH plays an important role in the removal mechanism. One of these lipid regulators, clofibric acid, is a common metabolite of clofibrate, and is detected frequently in the aquatic environment. Interestingly, research suggests clofibric acid concentrations increased at bank filtration sites in Germany due to the high consumption of liquid regulators during the 1990s. They discovered clofibric acid present in deeper layers of the aquifer. Lipid regulators and NSAIDs can be included in a joint research to test pH conditions for their removal. Steroid hormones are also a very particular group of CECs because they can produce potentially adverse effect on human health and aquatic life even at very low concentrations. However, laboratory scale and field studies using bank filtration in Berlin, Germany showed positive results. 17β-estradiol and 17α-ethinylestradiol were not detected in surface water from Berlin and estrone was removed greater than 80%. Snyder et al. used batch experiments and field studies with bank filtration and demonstrated estrone, 17β-estradiol, and 17α-ethinylestradiol were removed by biodegradation and sorption.Given conditions of certain MAR, steroid hormones and potentially a wide range of endocrine disrupting compounds can be reliably treated. For pesticides/herbicides, most studies have been focused on atrazine.Atrazine is resistant in the environment and penetrates through the surface and subsurface due to its high mobility, persistence, low vapor pressure, and massive application since it has been in use since 1959. Ho et al. and Zhang et al. showed triazine herbicides like atrazine were poorly removed by biofilters, but some studies have shown that atrazine can be readily biodegradable in aquatic environments, with reported removal rates ranging from weeks to years. 

Based on the previous studies, it is apparent that emerging contaminants can be removed and have shown adequate removals with a variety of conditions; aerobic and anaerobic conditions, biological substrate feeds, bacterial community development, filter mediums, variety of physicochemical properties of contaminants, and type of leachate water filtered using a SSF system. However, despite the variance in studies, most research agrees that the general contaminant removal increases over time as filter matures. Now that available freshwater resources are continuously limited and increases in world population have raised the pressure on natural resources , water resource management have turned to water reclamation and reuse to sustain agricultural activities. Reclaimed water use is not only limited to agriculture but widely used in other purposes such as irrigating landscapes, nurseries and greenhouses, flushing toilets, and replenishing groundwater aquifers. In 2006, an estimated 9.8×106 m3 d -1 of treated municipal wastewater was used in the United States. Studies have documented the presence of many micro-contaminants such as pharmaceuticals and personal care products, pesticides, phenolicestrogens, surfactants, biocides, and disinfection by-products in reclaimed water. These contaminants may accumulate in the area irrigated by reclaimed water and may result in contamination of the soil and plants. Along with the combined use of pesticides in greenhouses, it is possible for receiving waters to receive a wide range of pollutants. This is especially true in rural areas where contaminated runoff from farmland contributes a significant proportion of the pesticide load. The overall objective of this study was to promote slow sand filtration columns as a cost effective engineered solution to treat emerging contaminants. Specifically we simulated a greenhouse irrigation system that contains emerging contaminant concentrations of 400 ng L -1 based on the range of literature values for concentrations of emerging contaminants found in reclaimed nonpotable wastewater , a concentration higher than the average to be able to adequately detect the compounds in our study. This pilot scale study is a preliminary study to see how viable SSF columns can be to remove emerging contaminants. Results of the present study can be combined with previous studies of using SSF columns to remove both pathogens and contaminants provided by reclaimed water and pesticide use. The removal efficiencies of 14 selected pharmaceuticals and personal care products , 7 pesticides, 3 plasticizers, and 2 detergents/emulsifers, and the trends after the project’s initial start and declogging maintenance removal rates thereafter were examined. Chemical residues have been ubiquitous in the environment as they are found in many environmental matrices, from sewage water, effluent water from wastewater treatment plants, river water, to even drinking water. These compounds can come from sources such as households, nurseries, wastewater treatment plants, factories, hospitals, and any other facility dispensing chemical waste such as pharmaceuticals, personal care products, antibiotics, plasticizers, pesticides, etc. This group of compounds, known collectively as “emerging compounds” or “compounds of emerging concern” , is notorious for their occurrence in the environment and their complexity existing in particular environmental matrices. Even at their low concentrations they can affect human health and environmental health. Analytical techniques using Gas Chromatography-Mass Spectrometry or Liquid Chromatography-Mass Spectrometry have paved the way for determining the concentrations of CECs even at their trace amount. GC-MS was first used to determine pharmaceuticals and personal care products in the environment in 1976. Advances to the quantification of CECs in environmental samples have increased detection sensitivity and reliability. CECs come in a wide variability in their concentrations, polarities, and thermal labilities. 

Reconfiguration is used extensively in Corfu, to replace failed drives, to add capacity to the system, and to add, remove, or relocate replicas. This makes reconfiguration latency a crucial metric for our system. Recall that reconfiguration latency has two components: sealing the current con- figuration, which contacts a subset of the cluster, and writing the new configuration to the auxiliary. In our experiments, we conservatively seal all drives, to provide an upper bound on reconfiguration time; in practice, only a subset needs to be sealed. Our auxiliary is implemented as a networked file share.We now demonstrate the performance of Corfu-Store for atomic multi-key operations. Figure 3.19 shows the performance of multi-put operations in Corfu-Store. On the x-axis, we vary the number of keys updated atomically in each multi-put operation. The bars in the graph plot the number of multi-puts executed per second. The line plots the total number of Corfu log appends as a result of the multi-put operations; a multi-put involving k keys generates k +1 log appends, one for each updated key and a final append for the commit record. A client appending to a materialized stream first obtains the current layout and makes a request to the sequencer with a stream id. The sequencer returns both alog token, which is a pointer to the next address in the global log, and a stream token, which is a pointer to the next address in the stream. Using these tokens and the layout, the client determines the set of replicas to write to. In contrast to traditional designs, replica sets in vCorfu are dynamically arranged during appends. For fault tolerance, each entry is replicated on two replica types: the first indexed by the address in the log ,best vertical garden system and the second by the combination of the stream id and the stream address. To perform a write, the client writes to the log replica first, then to the stream replica.

If a replica previously accepted a write to a given address, the write is rejected and the client must retry with a new log token. Once the client writes to both replicas, it commits the write by broadcasting a commit message to each replica it accessed. Replicas will only serve reads for committed data. The write path of a client, which takes four round trips in normal operation is shown in Figure 4.5. A server-driven variant where the log replica writes to the stream replica takes 6 messages; we leave implementation of this variant for future work. The primary benefit of materialized streams is that they provide an abstraction of independent logs while maintaining a total global order over all appends. This enables vCorfu to support atomic writes across streams, which form the basic building block for supporting transactions. To append to multiple streams atomically, the client obtains a log token and stream tokens for each materialized stream it wishes to append to. The client first writes to the log replica using the log token. Then, the client writes to the stream replica of each stream. The client then sends a commit message to each participating replica. The resulting write is ordered in the log by a single log token, but multiple stream tokens. Materialized strreams are a first class abstraction in vCorfu, unlike streams in Tango which are merely tags within a shared log. Materialized streams strike a balance that combines the global consistency advantages of shared logs with the locality advantages of distributed data platforms. Specifically, the following properties enable vCorfu materialized streams to effectively support state machine replication at scale: The global log is a single source of scalability, consistency, durability and history. One may wonder, why have log replicas at all, if all we care to read from are materialized streams? First, the global log provides a convenient, scalable mechanism to obtain a consistent snapshot of the entire system. This can be used to execute long running read-only transactions, a key part of many analytics workloads, or a backup utility could constantly scan the log and move it to cold storage. Second, the log provides us with a unique level of fault tolerance – even if all the stream replicas were to fail, vCorfu can fall back to using the log replicas only, continuing to service requests.

Materialized streams are true virtual logs, unlike streams. Tango streams enable clients to selectively consume a set of updates in a shared log. Clients read sequentially from streams using a read Next call, which returns the next entry in the stream. Tango clients cannot randomly read from anywhere in stream because streams are implemented using a technique called backpointers: each entry in a stream points to the previous entry, inducing a requirement for sequential traveral. Materializing the stream removes this restriction: since clients have access to a replica which contains all the updates for a given stream, clients can perform all the functions they would call on a log, including a random read given a stream address, or a bulk read of an entire stream. This support is essential if clients randomly read from different streams, as back pointers would require reading each stream from the tail in order. vCorfu avoids backpointers, which pose performance, concurrency and recovery issues. Back pointers can result in performance degradation when concurrent clients are writing to the log and a timeout occurs,vertical farming equipment causing a hole filling protocol to be invoked. Since holes have no back pointers, timeouts force a linear scan of the log, with a cost proportional to the number of streams in the log. Tango mitigates this problem by keeping the number of streams low and storing multiple back pointers, which has significant overhead because both the log and the sequencer must store these back pointers. Furthermore, back pointers significantly complicate recovery: if the sequencer fails, the entire log must be read to determine the most recent writes to each stream. vCorfu instead relies on stream replicas, which contain a complete copy of updates for each stream, resorting to a single back pointer only when stream replicas fail. Sequencer recovery is fast, since stream replicas can be queried for the most recent update. Stream replicas may handle playback and directly serve requests. In most shared log designs, clients must consume updates, which are distributed and sharded for performance. The log itself cannot directly serve requests because no single storage unit for the log contains all the updates necessary to service a request.

Stream replicas in vCorfu, however, contain all the updates for a particular stream, so a stream replica can playback updates locally and directly service requests to clients, a departure from the traditional client-driven shared log paradigm. This removes the burden of playback from clients and avoids the playback bottleneck of previous shared log designs. Garbage collection is greatly simplified. In Tango, clients cannot trim streams directly. Instead, they must read the stream to determine which log addresses should be released, and issue trim calls for each log address, which can be a costly operation if many entries are to be released. In vCorfu, clients issue trim commands to stream replicas, which release storage locally and issue trim commands to the global log.While the Corfu log provides a scalable fabric for consistency, programmers need more than just a shared log abstraction to write reliable distributed programs. This chapter introduces the rich, object-oriented data services provided by Corfu. Instead of forcing programmers to produce and consume entries on the log, the distributed objects provided by Corfu enables applications to use the log by interacting with in-memory objects which are similar to common in-memory data structures used today. In addition to supporting in-memory objects, Corfu also supports transactions, which enable applications to access and modify multiple objects consistently. Finally, materialized streams enable local playback of the log, which enable thousands of clients to consume the log simultaneously.A Corfu application is typically a service running in a cloud environment as a part of a larger distributed system, managing metadata such as indices, name spaces, membership, locks, or resource lists. Application code executes on clients and manipulates data stored in Corfu objects, typically in response to networked requests from machines belonging to other services and subsystems. The local view of the object on each client interacts with a Corfu runtime, which in turn provides persistence and consistency by issuing appends and reads to an underlying shared log. Importantly, Corfu run times on different machines do not communicate with each other directly through message-passing; all interaction occurs via the shared log. Applications can use a standard set of objects provided by Corfu, providing interfaces similar to the Java Collections library or the C++ STL; alternatively, application developers can roll their own Corfu objects.The code for the Corfu object itself has three main components. First, it contains the view, which is an in-memory representation of the object in some form, such as a list or a map; in the example of a Corfu Register shown in Figure 5.1, this state is a single integer. Second, it implements the mandatory apply upcall which changes the view when the Corfu runtime calls it with new entries from the log.

The view must be modified only by the Corfu runtime via this apply upcall, and not by application threads executing arbitrary methods of the object. Finally, each object exposes an external interface consisting of object-specific mutator and accessor methods; for example, a CorfuMap might expose get/put methods, while the Corfu Register in Figure 5.1 exposes read/write methods. The object’s mutators do not directly change the in-memory state of the object, nor do the accessors immediately read its state. Instead, each mutator coalesces its parameters into an opaque buffer – an update record – and calls the update_helper function of the Corfu runtime, which appends it to the shared log. Each accessor first calls query_helper before returning an arbitrary function over the state of the object; within the Corfu runtime, query_helper plays new update records in the shared log until its current tail and applies them to the object via the apply upcall before returning.Based on our description thus far, a Corfu object is indistinguishable from a conventional SMR object. As in SMR, different views of the object derive consistency by funneling all updates through a total ordering engine. As in SMR, strongly consistent accessors are implemented by first placing a marker at the current position in the total order and then ensuring that the view has seen all updates until that marker. In conventional SMR this is usually done by injecting a read operation into the total order, or by directing the read request through the leader; in our case we leverage the check function of the log. Accordingly, a Corfu object with multiple views on different machines provides linearizable semantics for invocations of its mutators and accessors. A Corfu object is trivially persistent; the state of the object can be reconstructed by simply creating a new instance and calling query_helper on Corfu. A more subtle point is that the in-memory data structure of the object can contain pointers to values stored in the shared log, effectively turning the data structure into an index over log-structured storage. To facilitate this, each Corfu object is given direct, read-only access to its underlying shared log, and the apply upcall optionally provides the offset in the log of the new update. For example, a CorfuMap can update its internal hash-map with the offset rather than the value on each apply upcall; on a subsequent get, it can consult the hash-map to locate the offset and then directly issue a random read to the shared log. Since all updates are stored in the shared log, the state of the object can be rolled back to any point in its history simply by creating a new instance and syncing with the appropriate prefix of the log. To enable this, the Corfu query_helper interface takes an optional parameter that specifies the offset at which to stop syncing.

A recent analysis of pit latrines concluded that globally, pit latrines accounted for 1% of anthropogenic CH4 emissions. The relatively large contribution of pit latrines to global CH4 sources can be attributed to the global extent of pit latrine use e approximately one-quarter of the global population e as well as the wet and unventilated conditions that drive anaerobic CH4 production. EcoSan relies on aerobic conditions to treat waste and has the potential to considerably reduce the GHG footprint of waste management. In aerobic thermophilic composting, CH4 emissions are typically low because of the presence of oxygen. However, anaerobic microsites created by uneven distribution of water in pores and hot spots of labile carbon can create conditions leading to CH4 emissions. The use of bulking agents and pile turning can be used to reduce the occurrence of anaerobic CH4-producing conditions and, when effective, carbon emissions from composting are in the form of CO2, which is considered to be climate-neutral because of its biogenic origin. Composting can, however, produce biogeochemical conditions prime for N2O emissions through nitrification or denitrification, including large sources of reactive nitrogen, dynamic and spatially varying levels of oxygen, and labile carbon sources. Quantifying the magnitude and balance of CH4 and N2O emissions in a given sanitation system is critical as the two gases have 100-year global warming potential values of 34 and 298, respectively. EcoSan systems utilizing aerobic, thermophilic composting are promising because they may mitigate GHG emissions from the waste and agricultural sectors, however these emissions reductions have not yet been quantified. Further, measurements of GHG emissions from management of solid organic wastes are especially limited from tropical climates , vertical farm system where implementation of EcoSan solutions are likely to be greatest.

To our knowledge, no direct measurements of GHG emissions exist from EcoSan systems that deploy container-based toilets and thermophilic composting of human excrement. Our primary objective was to characterize and quantify the GHG emissions resulting from the aerobic composting of human waste in EcoSan settings. We considered two operations that employed similar compost practices, but differed in the physical infrastructure that could alter biogeochemical conditions mediating GHG dynamics. We also compared the GHG footprint of EcoSan with alternative waste management pathways present in the region, including waste stabilization ponds and unmanaged disposal on grass fields. Finally, we undertook an investigation of the effects of compost management options that help reduce EcoSan GHG emissions.Greenhouse gas fluxes were measured from three sanitation pathways in Haiti: two waste stabilization ponds, two EcoSan operations, and a grass field where the illegal disposal of sewage was observed. The waste stabilization ponds were located in Croix ed Bouquets near Port-au-Prince, Haiti and operated by the Haitian government agency, Direction Nationale de l’Eau Potable et de l’Assannissement. Ponds consisted of uncovered concrete basins with effluent pipes connected to secondary overflow ponds. Two ponds were included in the sampling: a pond that received mostly septic tank waste , and another that received mostly pit latrine waste. Solid sludge was scraped out occasionally and stockpiled on-site. Solid and liquid waste from septic tanks and pit latrines were transported to the site and emptied into the waste stabilization ponds. The waste stabilization ponds represent the primary pathway of centralized waste treatment as advanced municipal wastewater treatment technologies are not present in the country. The EcoSan compost facilities were located in Port-au-Prince, Haiti and Cap Haiti€ en, Haiti. The sites received approximately 65 MT yr 1 and 440 MT yr 1 of human waste, respectively. Eighteen and 54 L container-based toilets were collected from households and communities, respectively, in each of the regions. Container-based toilets separated urine and feces into different compartments.Urine was disposed of on-site, and only solid material was transported to the compost facilities. Both facilities used a similar aerobic composting process consisting of a static thermophilic stage, followed by pile turning and maturation in windrows.

At the Cap-Haiti€ en operation, hereafter referred to as “Compost CH,” the ground was lined with cement to prevent leaching and an aluminum roof covered the area. Roofs and cement-lined floors were absent at the Port-au-Prince EcoSan operation, hereafter referred to as “Compost PAP”. During the initial thermophilic stage, approximately 2700 kg of fresh material from container-based toilets was added to a ~27 m3 bin consisting of air-permeable walls and an open top. Coarsely ground sugarcane bagasse was used as a bulking agent to create interstitial air spaces within the pile and as a 15 cm deep covering material. The material remained in the bin for about two months or until confirmation of Eschericia coli elimination, during which time it underwent static, thermophilic composting, reaching a peak minimum temperature of 50 C for at least 7 days. Following the thermophilic stage, the material was removed from the bin onto a flat surface, formed into windrows and aerated by weekly manual turning for two to three months. Finally, matured compost was then sieved and bagged for use as a composted soil amendment. The third waste disposal pathway was an unmanaged grass field near Quartier Moren, Haiti. Unregulated emptying of septic and pit latrine waste is not uncommon and has been observed here for at least five years and within a few months of sampling. While the frequency and magnitude of waste disposal in the grass field was unknown, there was apparent build up of organic material that resembled a moist, viscous sludge several inches to feet deep.Greenhouse gases were measured once at each site within seven days in July 2014. Fluxes of CO2, CH4, and N2O were measured using vented static flux chambers constructed of 25.4 cm diameter Schedule 80 PVC collars and chamber tops. 1.5 m tall collars were placed carefully in the semi-solid sludge in the waste stabilization ponds,vertical indoor farming approximately 1 m from the edge.Four areas were sampled in Pond 1, and six areas were sampled in Pond 2. Gas samples collected within ponds were treated as replicates and used to determine mean fluxes from each pond. Six 0.3 m tall collars were placed randomly in the Grass Field and allowed to settle for 1 h before the chamber tops were connected. Gas measurements were also made within and across compost piles of different stages. While sampling was conducted at each site at one time point, compost piles exist along a gradient of ages from freshly collected waste to mature compost. This design allows us to effectively substituting space for time to determine mean flux from each EcoSan system over the entire composting process.

At Compost PAP, eight piles ranging from <1, 1, 2, 4, 6, 8, 10, and 13 months were sampled, with six static flux chambers randomly placed in each pile. At Compost CH, six piles ranging from <1, 3, 4, 5, and 12 months old were sampled, with three static flux chambers randomly placed in each pile. Linear interpolation between age classes was used to calculate the net GHG emissions from EcoSan compost operations. Mean GHG emissions were determined by weighting fluxes by age of the pile. Gas samples were collected from the static flux chamber head space at 0, 5, 10, 20, and 30- minute intervals, immediately transferred to evacuated 20-mL Wheat on glass vials outfitted with 1-inch butyl septa. Samples were transported to the Cary Institute of Ecosystem Science in Millbrook, New York for analysis on a gas chromatograph. Methane concentrations were analyzed using a flame ionization detector. An electron capture detector was used to analyze N2O concentration, and CO2 concentrations were analyzed using a thermal capture detector. Samples with concentrations exceeding the maximum detection limit on the gas chromatograph were diluted at 1:10 or 1:100 with N2. Fluxes were calculated using an iterative exponential curve- fitting approach.To explore how compost management impacts GHG emissions, we established an additional experiment at the Compost CH site in August 2016. First, to test the effects of pile lining permeability, we measured GHG emissions using the procedure described above from a bin with a soil floor and from a bin with a cement floor and a blocked PVC overflow pipe , which were filled at approximately the same time. Second, to test the effects of pile turning, we measured GHG emissions one and three days after unlined bin material was turned for the first time, and compared emissions to those before turning. Twelve fluxes spaced evenly on a four x three grid were measured for each bin in the first stage to explore spatial variability of GHG emissions within the pile.

Six fluxes were measured per pile in the second turned stage, spaced on a three x two grid. The grid design also allowed us to explore the effects of pile structure and geometry on GHG emissions in greater detail.One-way analyses of variance followed by TukeyKramer means comparison tests were used to identify statistically significant effects of waste treatment pathway on mean CO2, CH4, and N2O fluxes. Treatment fluxes sampled across waste pathways were calculated as the mean and standard error of replicate samples collected within each system. Standard errors were propagated when considering mean GHG fluxes throughout the entire compost process at the EcoSan sites. For the compost management experiments, fluxes are represented as the mean and standard error of sample replicates. Fluxes of each gas species were considered separately and in combination using units of CO2-equivalents, using the 100-year global warming potential of 298 for N2O and 34 for CH4. Gas flux data were log-transformed to meet assumptions for ANOVA. Data are reported either as mean values±one standard error. Statistical significance was determined as P < 0.05 unless otherwise noted.We found pile lining permeability and pile turning significantly altered GHG emissions during EcoSan composting. A permeable soil lining lowered GHG emissions with pile CH4 and N2O emissions four and three-fold lower, respectively, in the unlined pile compared to the cement lined pile. Emissions also followed spatial patterns within the piles with CH4 emissions generally increasing from corners and edges to the center of the pile, with the opposite trend observed for N2O. Finally, substantial changes were observed in all GHGs after pile turning: CO2 and N2O emissions approximately doubled while CH4 emissions dropped almost to zero.We found that composting human fecal matter resulted in significantly lower overall GHG emissions than those observed from waste stabilization ponds and the illegal disposal of human waste on unmanaged grass fields. In EcoSan systems, CO2 was a major constituent of the overall GHG footprint, making up 42e62% of net CO2-eq emissions. In contrast, CO2 made up only 12% from grass fields used for dumping of untreated waste and 4e9% of the net CO2-eq emissions from waste stabilization ponds, suggesting that the ponds are not completely anaerobic. The difference in CO2 loss is likely driven by differences in oxygen availability among the waste treatment pathways. Composting systems enhance aerobic conditions that stimulate the microbial oxidation of organic carbon to CO2 , whereas biological treatment systems with oxygen-depleted conditions stimulate methanogenesis and production of CH4. Carbon dioxide produced from the decomposition of human waste is of biogenic origin, and therefore not considered a net source of GHG emissions from a climate change perspective. Conditions that evenly aerated compost piles, such as forced aerated and pile turning, tend to maximize CO2 production relative to CH4 and N2O. However, compost piles tend to have high levels of bio-geochemical heterogeneity due to within pile spatial variations in organic compounds, physical size and structure of material, oxygen diffusivity, density, porosity, and moisture content. Therefore, compost pile management can play a strong role in altering biogeochemical conditions that effect the composition of GHG fluxes. In the two EcoSan systems that we studied, we saw significantly higher CH4 fluxes from Compost CH, where static piles sat atop a concrete floor during the thermophilic phase. The presence of the floor likely built up moisture in the pile and increased the probability of anaerobic microsites. Methane emissions from Compost CH rapidly declined after the first two to three months, when piles were moved into the actively turned stage. In contrast, compost piles at Compost PAP were constructed atop compacted soil and without a roof covering, allowing for infiltration of leachate and higher rates of evaporation.

The original proportion of amorphous and crystalline cellulose before and after milling and 30–50% of sample crystallinity was lost after milling cotton for 15 minutes.Based on 1D CP solid state NMR measurements around 40% of crystallinity was lost and the initial crystallinity of the sample accounted for some inherent amorphous cellulose compared to x-ray scattering techniques.When pure cotton cellulose was milled crystalline cellulose fibrils showed loss in crystallinity and a consistent increase in amorphous cellulose, supporting mechanical preprocessing could induce recalcitrance.To test the current mechanical induced recalcitrance hypothesis in mechanical preprocessing, sorghum stems were subjected to lab scale vibratory ball-milling.In this study, stem tissue was used for initial experiments to allow for the highest concentration of secondary plant cell wall to increase the likelihood of observing recalcitrance with the greatest signal-to-noise ratio possible for solid-state NMR.Monitoring changes in sample morphology of the cellulose fibrils by FE-SEM is necessary because milling times vary depending on the biomass, sample quantity, and desired particle size.The time points include milling for 2 minutes at 30 Hz as common initial mechanical preprocessing time consistently executed for biomass18 and 15 minutes of milling at the same frequency to emulate longer milling times.Contrasting the effects of milling pure cellulose and milling cellulose in the plant cell wall also informed the selection of the 15-minute milling time point.Molecular changes in secondary plant cell wall polymers are probed with an initial set of solid state NMR experiments.The rINEPT was expected to selectively probe the highly dynamic polymers, lignin, and hemicellulose.

The CP-rINADEQUATE 66 probes directly bonded carbons in rigid components such as cellulose of the cellulose fibril and hemicellulose associated to cellulose fibrils.The CP-PDSD experiments were used to monitor the atomic cellulose morphologies using through space carbon-carbon correlations.Molecular recalcitrance markers include mobility and contacts of the dominant polymers by solid-state NMR experiments for polymers lignin, hemicellulose,vertical lettuce tower and cellulose.Monitoring markers of recalcitrance can be subdivided into each type of polymer.During the milling process crystalline cellulose is expected to convert to amorphous cellulose according to previous work on pure cellulose fibril structures of cotton by 1D NMR, FE-SEM, and vibrational spectroscopy.The resulting conformation change to amorphous cellulose within the plant cell wall may result in decreased access to crystalline planes of cellulose necessary for enzyme digestion.Assuming sample matter is conserved and no digestion occurs, proportions of crystalline and amorphous cellulose content in cellulose fibrils can be reported by NMR.5 Unlike vibrational spectroscopy where there is high signal overlap and sample crystallinity indexes afforded by x-ray diffraction, NMR contains well separated signatures for amorphous and crystalline cellulose.Cellulose polymers on the dehydrated interior, center, and more hydrated interior cellulose polymers of the cellulose fibril have distinguished chemical shifts over the last 20 years of experiments and specifically identified for sorghum.24 In 2D solid-state NMR experiments plant cell wall polymers compromise over a third of biomass and most of the signals due to the 13C labeling technique.When examining the plant cell wall in the spectra the carbon neutral region of polys accharides and lignin is 120–60 ppm and 170–110 ppm, respectively.Tracking proportional intensity changes of amorphous and crystalline cellulose distinguished by their chemical environments is possible.

Cross Polarized NMR experiments will select for the rigid polymers as the experiments are able to spectroscopically separate the rigid portions of the plant cell wall including the cellulose fibrils.CP based solid-state NMR experiments are more efficient for spins stagnant in spacepolymers of the plant cell wall.Cellulose fibrils are solid structures which can be probed with CP based solid-state NMR.Hemicellulose rigidly associated to cellulose in cellulose-hemicellulose interactions would also be selected for using CP experiments.In the context of the plant cell wall, cellulosehemicellulose interactions do vary based on the morphology of cellulose,so recalcitrance could also occur by additional amorphous cellulose-hemicellulose interactions common in grasses such as sorghum.Hemicellulose signals would then increase in their rigid signal intensity according to CP experiments and decrease in signal intensity from the highly dynamic signals.The highly dynamic signals captured in the rINEPT include 1H-13C correlations in the 2-D experiment and lignin with the plant cell wall.Lignin reorganization could cause recalcitrance in a variety of ways.One observable way would be cross-linkages forming due to mechanical activation ligninarabinose-xylan.The ether signals within lignin branching and lignin-hemicellulose cross-linkages are captured within the rINEPT experiment.Lignin condensation, aggregation, and cross linking noted in recalcitrance means highly dynamic lignin content is lowered , resulting in an expected decrease in signal intensity in the rINEPT.Digestible hemicellulose and cellulose would be reduced as polys accharides are trapped as lignin condenses in the secondary plant cell wall.Based on previous 13C evident methods and solid-state NMR analysis, the molecular evaluation of secondary plant cell wall architecture is feasible for bio-product relevant crops such as sorghum.This research project aims to identify where recalcitrance occurs in the deconstruction pathway and identify markers of recalcitrance using 2D solid-state NMR.The first step of the deconstruction pathway is investigated for mechanically induced recalcitrance for 13C labeled sorghum.How the heterogeneous secondary plant cell wall changes from preprocessing needs to be carefully approached.This work primarily focuses on intensity changes of unambiguous signals within the established sorghum native plant cell wall to circumvent misassignment of polysaccharide non-ambiguous peak changes which shift to ambiguous, overlapped chemical shifts during deconstruction.

Gao et al.2020 is used as a reference for secondary plant cell wall polymer chemical shift assignments confirmed in sorghum from previous literature which relied on extracted polymers,computational verification,and previous solid-state NMR9,on the architecture of the plant cell walls.Additional structural information can be confirmed from chemical shifts using computation, allowing for polymer morphological changes to be assessed based on changes in monomer orientation.Future work using data collected in this work could offer greater insight into polymer morphology changes.Given the heterogeneity of the plant cell wall samples,vertical grow rack system examining the recalcitrant markers would narrow the relevant chemical shift and chemical shift changes by computation as well as contrast chemical shifts of extracted polymers.Chemical shifts can support changes in polymer environments when peaks are well dispersed and unambiguous within a 0.5 ppm.For example, accessible polymers are expected to be more hydrated and have chemical shifts downfield.As cellulose fibrils are broken down into more disperse micro-fibrils or cellulose substructures, as discussed in Figure 2C, the chemical shifts of amorphous and crystalline cellulose may be expected to move downfield.Additional hemicellulose-cellulose association on hydrophilic surfaces of cellulose polymers would have chemical shifts potentially shift upfield for both polysaccharides.Upon the hemicellulose crosslinking to lignin may also result in chemical shift changes downfield for hemicellulose due to deshielding from bonding with the conjugated heteroaromatic lignin.However, recalcitrant organization of lignin trapping hemicellulose and cellulose in self-aggregation would reduce surface water may have chemical shifts moderately upfield compared to the expected literature values in sorghum based on Gao et al.2020 assignments of the secondary plant cell wall.These are generally anticipated chemical shift changes in polymers which is somewhat challenging given signal overlap of polysaccharides.In this work changes in chemical shift after mechanical preprocessing are highlighted by 2D integration of chemical shifts found in the control.Integration allows for minor changes or gradual changes in the plant cell wall to be assessed, increasing the ease of interpreting reasonable chemical shift changes upon preprocessing.Sorghum was grown hydroponically and then transferred to a growth chamber for carbon dioxide 13C labeling until they reached 16 weeks by collaborators Yu Gao and Jenny Mortimer as described in Gao et al.2020.The sorghum was incorporated with 92% 13C according to elemental analysis and are physiologically normal organisms.Upon harvest, the plants were flash frozen in liquid nitrogen, divided by tissue type , and cryogenically stored for later studies.

Samples were transported on dry ice between labs and stored at −80 °C for the duration of the study.Cut stems were milled using a Retch MM400 in a 10 mL zirconium grinding jar with two 10 mm zirconium beads at 30 Hz.For the 2-minute milling time point, 600 mg of cut stems were milled for a single 2-minute interval.For the 15 minute milling period, 600 mg of cut stems were milled in cycles for 5 minutes followed by 5 minute resting periods in accordance to relevant lab scale milling procedures from previous literature.After the milling period, the samples were divided into several cryogen vials and flash frozen in liquid nitrogen before storage in the −80 °C freezer.Sample Morphology Tracked with FE-SEM FE-SEM was employed to check sample morphology, specifically cellulose fibril morphology structures after milling.A Hitachi S-4100 T Scanning Electron Microscope was used to collect high resolution images for the control and milled samples using a procedure detailed in Zheng et al.2020.98 A 10 nm gold coating was sputter coated onto control and milled sorghum samples with a Cressington 208hr Sputter Coater.FE-SEM images were collected at a working distance of 15–6 mm with acceleration voltages ranging from 2–10 kV to achieve optimal spectra resolution.Atomic Resolution of Sorghum Stem Secondary Plant Cell Wall by MAS-ssNMR NMR experiments were performed on a Bruker 500 Solids NMR Spectrometer , with a Bruker 4 mm MAS probe and at a MAS speed of 10 kHz at the UC Davis NMR Facility.All samples were shimmed using a water sample file on the instrument.Two channels were utilized on the 500 MHz instrument, one was set to SFO2 500.0305 MHz for the proton and the other was set to 125.7445782 MHz for 13C.Spinal 64 1H decoupling was used.Decoupling powers were optimized for 1H radio frequencies of 71 kHz.The radio frequency power for CP on the carbon for the 1D and 2D NMR was a matched between protons and carbons at the 10 kHz MAS spinning frequency.32 Optimization of the 1H and 13C RF pulse lengths for each sample were obtained by locating nulls a carbonyl signal around 100 ppm in a 1D CP experiment with 4 scans with parameters similar to the 1D CP in Table 1.An 1-13C L-alanine sample was used to check instrumentation and calibrate chemical shifts on the TMS scale using a 1D CP experiment prior to data collection on the plant samples.99 Beyond these commonalities, parameters for the experiments applied to all the control and milled samples are summarized in Table 1.Samples were packed into 4 mm zirconium rotors with two 2 mm Teflon discs cut from Teflon tubing on the top and bottom to center the 13C sample.All data collection occurred at room temperature.Experiments on control stems revealed the total time of sample viability in the spectrometer to be approximately 52 hours and this was tracked for all experiments by periodic the collection of 1D CP and DP spectra for all sample data collections.The 2D was collection is divided into two sets of experiment collection per experiment as summarized in Table 1 and added together in NMR pipe for processing.NMR data were processed in NMRpipe with Gaussian line broadening applied before the Fourier Transform.100 Spectra were plotted in Sparky101 and formatted in Corel Draw 2019.A quantitative 1D DP experiment was employed to properly scale data on the milled and control samples for integration.Parameters of the quantitative 30 s 1D DP experiments were like 1D-DP experiment in Table 1 but the recycle delay was adjusted to 30 second recycle delay to assess the 13C content within the control and the milled stem samples.After data collection samples were stored at −80 °C in their respective 4 mm rotors for later use in the 1D-DP to quantitatively scale the sample loading of the samples which varied in consistency.The 30 s 1D-DP allowed for integrations across all experiments for each sample to be scaled for sample load which varied due to the consistency variation in the cut and milled samples.Milled samples were flash frozen before NMR data collection without changes to the plant cell wall.Cut stems were thawed for 3.5 hours based on the appropriate milling time for DMSO gel swelled plant cell walls of grasses outlined for 600 mg of material in Kim and Ralph 2010.Experiments in Table 1 and a DP-rINADEQUATE show no detected changes.The DP-rINADEQUATE had the same experimental parameters outlined in the CP-rINADEQUATE summarized in Table 1 with the cross polarization removed and the 13C nuclei directly irradiated.This meant samples could be milled, flash froze, and rethawed for data collection.

The plain hosts shallow water tables, which, combined with negative climatic water balances, makes it prone to salt accumulation both in its deep sediments and in the surface of its low landscape sectors.Grasslands dominate in the South of the sedimentary plain, showing some areas with salt affected soils.Besides those areas also few coastal and swampy areas with saline-acid soils are found as well as large internal salt marshes elsewhere, among them the Pantanal in southern Brazil, one of the larger wetlands of the world.Overview of anthropic salinity problems—In general terms, no country in Latin America is completely free from salinization, and we will focus our analysis on human-induced salinization, mainly caused by irrigation, though not exclusively.Most of the secondary salinization occurs in irrigated areasin arid and semi-arid zones, where intensive agriculture is practiced.This process is mainly due to non-efficient water management, poor drainage conditions, and low irrigation water quality.Irrigation mode and extension as well as type of crops vary considerably within the region.Fruit and vegetable production are mostly irrigated.In some area’s extensive crops such as sugarcane, rice, cotton, maize, and wheat are partially grown under irrigation, using modern technologies.In others, irrigated areas are populated by small holder farmers who generate most of the locally consumed food.The ratio between these two ways of production varies among countries and regions within them but does not to appear to be related to soil salinization processes.Arid and semi-arid areas under full irrigation are common in Mexico, Peru, Chile, and Argentina.While irrigated production value exceeds that of rainfed agriculture in Mexico, Chile and Peru, the reverse occurs in Argentina.In most cases furrow and flooding irrigation systems are used, but sprinkler, micro-sprinkler or drip irrigation systems are being increasingly adopted.

Waters from various origins are used,vertical farming tower for sale from surface to ground waters, and quality ranges from good to bad.Irrigation in non-graded and uneven lands has led to low water use efficiencies and rising groundwater levels.Low efficiencies are additionally caused by non-lined water distribution ditches and poor drainage conditions.Besides the typical effects of salts , boron is an additional problem in several irrigation districts.The Brazilian semiarid region in the northeast of the country is one of the largest semiarid regions of the world.It features tropical climate conditions with variable rainfall associated with high temperatures during much of the year.The region is also characterized by shallow soils, low quality irrigation waters, lack of drainage and often shallow groundwater.Irrigation has improved the economy of the region through diversified cropping practices, stimulation of agroindustry and export of products, but has also led to large areas being degraded by salinization because of poor water quality and deficient or absent drainage schemes.In general, those degraded areas are left fallow so that agricultural production is moved to other areas.The return of vegetation of these deserted areas would initiate a slow reclamation process.In semiarid/subhumid zones, as in Colombia, Venezuela, Cuba, the Dominican Republic and in some of the other countries in the region, similar salinization processes have occurred where sugarcane, rice and othertropical crops are irrigated with waters of varied quality and drainage is poor or nonexistent.Further south, in the temperate area of Pampas region of Argentina, field crops are usually grown under rainfed conditions but are exposed to occasional drought events.Supplementary sprinkler irrigation allows farmers to increase and stabilize grain yields.Exchangeable sodium has increased sharply but no consistent impacts on soil physical degradation have been detected.Human-induced salinization has also developed in the above-mentioned Chaco-Pampas plain, mainly promoted by land use and land cover changes, by cropping or overgrazing.In the Chaco and Espinal regions in the North of this plain, deforestation and cultivation have altered the hydrologic balance, mainly because cropped areas present lower evapotranspiration rates.

The resultant water excess infiltrates and slowly causes the rise of deep groundwater tables that bring salts to the surface, thereby damaging crops and soils.This process of salinization is somewhat like the “dryland salinization” in Australia.The Southern part of this Chaco-Pampas is mostly devoted to field crops, but alkaline and to a lesser extent saline soils predominate in an area known as the “Flooding Pampa,” where livestock production activities prevail.There, intensive cattle grazing removes vegetation and high evapotranspiration causes salts from the water table to reach the soil surface in the summer.Subsequent rains leach the salts, but this man-made process affects the composition of the plant communities.A similar process has been observed in grazed salt wetlands.Research on salt affected soils was very active in the 1960–1980 period, when several countries experienced large agricultural development through investments in large irrigation schemes.Research on soil salinity research at that time was mainly applied and based on results published by the US Laboratory Staff.One consequence of such effort was the organization of regional and international conferences, such as those that took place in 1971 in Colombia and in Venezuela in 1983.However, advances in research and evaluation of salt-affected soils subsequently faded.In most irrigated areas attention was focused on the engineering aspects of irrigation infra-structures rather than on the installation of effective drainage systems and adequate preparation of irrigation fields at the farm-scale.This has led to problems of drainage, water logging,and salinization.More recently, the development of large and expensive irrigation schemes has diminished, whereas new irrigation developments have been for small local irrigation units, using nearby surface and groundwater resources but most often done without consideration of regional impacts.In some extreme cases, due to competition for alternative uses of scarcely available good-quality water resources, non-treated residual waters of urban and industrial origin are used for irrigation.This is valid for small irrigation units, mainly dedicated to production for local markets, but not for larger irrigation units.Research on soil, water, and crop management in saline areas in Brazil is concentrated in universities and research organisms in NE Brazil.

Approaches include the development of soil and water management strategies, appropriate cropping systems, the sustainable use of brackish waters, cultivation of halophytes and salt-tolerant crops, application of mineral and organic amendments, phytoremediation and plant/microorganism interactions.A specific concern is the mitigation of socio-economic impacts of soil salinity in agricultural lands, which translate into loss or reduction of crop yields, profit margins, increased unemployment, and reduction of commercial land value.Technologies are being developed to provide a source of income for impacted smallholder farmers to provide for water and food security.This include the desalination of brackish water and its use in an integrated production system involving reject brine for farm-raised fish and the use of fish-pond water to grow organic salt-tolerant vegetables and forage crops for small ruminants.Argentina has active research on soil, water and crop management and salt tolerance mechanisms.Technologies on salt affected soils of humid/ sub-humid areas are aimed mainly at increasing biomass productivity without altering soil properties.They include grazing management, afforestation, agro-hydrological management, plant introduction, among others.Salinization in semiarid deforested areas has been studied in the great Chaco area and is focusing on ways to mitigate soil and water quality degradation , such as by changing cropping systems.The use and management of native woody species for degraded and salinized areas is considered.The characterization, collection and multiplication of both native and introduced species, and their incorporation into breeding program are prominent activities.Traditional breeding efforts have produced new salt-tolerant forage plant cultivars, such as Epica INTA Peman ,hydroponic vertical farming and new breeding alternatives have been explored to increase salt tolerance in Melilotus albus.Research on Lotus species for alkaline and sodic soils has contributed to their expansion in the Flooding Pampa.Molecular components of signaling chains and salt tolerance mechanisms have been successfully incorporated into commercial crops, soybean for example.Research interest in the region on salinity-related agricultural aspects has gained new momentum in this century, mainly in Brazil, Mexico, Argentina and Chile.In addition to many publications, this is also reflected by recent national salinity conferences in Argentina and in Brazil.The First Latin American Salinity Symposium was held in Fortaleza, Brazil, in 2019.Books on regional salinity issues have been published in Spanish and in Portuguese , including a recent comprehensive book.The subcontinent is re-awakening to its saline perspective.Social impacts of this problem are being addressed, particularly because of food security issues.The process of salinization in irrigated areas is continuing, although in some cases drainage and improved irrigation technologies improved the situation noticeably.However, in many Latin-American regions soil salinization is still expanding.Deforestation has been extensive, and the consequences of these land use changes will further cause land degradation and affect the sustainability of its land and water resources.It is expected that the coming decade will provide more certain quantification of its increasing spatial extent, as FAO and various organizations from Latin American countries are on the way to develop a contemporary soil salinization map, following unified protocols.

Problems with salinity in the Netherlands mainly occur in the North Sea coastal regions.Fig.31 shows in blue colors areas either above or below sea level protected by dikes and in orange color areas below sea level not protected by dikes.The white areas along the coast, including islands in the north and southwest, are dunes in which fresh water floats on sea water.Throughout history, various aspects of salinity were recognized and dealt with, specifically causes of salinization and sodification leading to soil structure deterioration, desalinization and rehabilitation by de-sodification, and crop salt tolerance/intolerance.Originally, experience of water managers and farmers formed the basis in their decision-making.From 1850 onwards, traditional opinions gradually evolved into scientific understanding.First, these were mainly based on chemical analysis of soils, later combined with physico-chemical concepts, and more recently through inclusion of analyses of flow and transport processes and plant physiology.In the first half of the 20th century, salinization and sodification arose from both natural floods and wartime strategic inundations.In the aftermath of the large 1916 flood, plans were made for the Zuiderzee Works, resulting in completion of the Afsluitdijkin 1932, changing the former tidal and saline Zuiderzee in a freshwater environment.Behind this dam are now the freshwater Lakes IJssel and Marken, surrounded by a series of new polders with a total area of 165.000 ha.In other words, where formerly was the Zuiderzee are now two lakes and the Wieringer meer and Flevo polders.The two lakes serve as fresh water reservoirs for the northern provinces, including replenishment of water pumped for domestic use in the coastal dunes.The early salinity research in the Netherlands was linked to consequences of the major floods and the Zuiderzee Works.Much of this specific research was presented in Raats and includes pioneering studies by Dutch scientists through the later 1800s and into the first half of the 20th century.Specifically, attention was paid to acid sulfate soils, application of gypsum to remedy soil structural degradation, analysis of plant salt tolerance, planting of salt tolerant vegetation to reclaim lands below sea-level , and understanding seepage from saline open water into lower lying land.Immediately following the devastating February 1953 Storm flood, the Delta Plan was launched, aimed at preventing recurrence of damage from such rare, huge storms in the future.The plan included upgrading all dikes along the entire coastline and build a series of barriers in the Southwest to close off all tidal inlets , except the Western Scheldt.The original main aims were protection of life and property and reduction of costs of maintenance of dikes.A reduction of saline seepage into many polders on the islands in the Southwest would also have resulted.During the execution of the Delta Plan, pressure from environmentalists and fisherman ultimately led to drastic changes in the plans.While construction of a dam in the Eastern Scheldt had started already in 1960, it was not until 1979 that parliament approved a novel type of storm surge barrier, with gates that can be closed when necessary.This barrier was completed in 1986.Earlier, in 1974 it was decided to keep the planned fresh water Grevelingen Lake saline by means of a sluice in the dam, which was completed in 1978.Density stratified flows—Already in the 1950s W.H.Van der Molen noted the occurrence of high salinities in the North-East Flevo Polder at depths of 10–15 m in places where a highly permeable Pleistocene deposit reached the land surface.He speculated that these high salinities were probably due to convection currents caused by the small difference in density between the freshwater present in the soil and the supernatant seawater of the former Zuiderzee between 1600 and 1931 AD.